LOCUS       AICSDP-36: AAVS1-mEGFP        7388 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Homology arms and linker-mEGFP sequence for internal insertion of 
            PGK-mEGFP at the AAVS1 safe harbor location in human cells as a 
            reporter for cytoplasmic mEGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7388)
  AUTHORS   Allen Institute for Cell Science
  TITLE     Allen Institute for Cell Science Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7388)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: AICSDP-36: AAVS1-mEGFP (PGK)
FEATURES             Location/Qualifiers
     source          1..7388
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    178..679
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    806..831
                     /label=SA
                     /note="splice acceptor site"
     CDS             855..908
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             918..1517
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(918..937)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1414..1434
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1549..1566)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1555..1779
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1871..2371
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2290..2308
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(2393..2395,2396..2398,2399..3112)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2438..2459)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2699..2718)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3046..3067
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3202..3221)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3267..3322
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3321..3340)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3683..3699)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3707..3723
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3731..3737,3738..3755,3756..3761))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3776..3797
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    3871..4707
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(4742..4758)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4766..4782
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4790..4796,4797..4814,4815..4820))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4835..4856
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4973..4990)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5144..5732)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5224..5243)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5903..6694,6695..6763))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6526..6545
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6764..6868)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6936..6954
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6992..7014)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7114..7133
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7327..7349
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7341..7358
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7342..7358
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tctctgacca gcattctctc ccctgggcct gtgccgcttt ctgtctgcag cttgtggcct
       61 gggtcacctc tacggctggc ccagatcctt ccctgccgcc tccttcaggt tccgtcttcc
      121 tccactccct cttccccttg ctctctgctg tgttgctgcc caaggatgct ctttccggag
      181 cacttccttc tcggcgctgc accacgtgat gtcctctgag cggatcctcc ccgtgtctgg
      241 gtcctctccg ggcatctctc ctccctcacc caaccccatg ccgtcttcac tcgctgggtt
      301 cccttttcct tctccttctg gggcctgtgc catctctcgt ttcttaggat ggccttctcc
      361 gacggatgtc tcccttgcgt cccgcctccc cttcttgtag gcctgcatca tcaccgtttt
      421 tctggacaac cccaaagtac cccgtctccc tggctttagc cacctctcca tcctcttgct
      481 ttctttgcct ggacaccccg ttctcctgtg gattcgggtc acctctcact cctttcattt
      541 gggcagctcc cctacccccc ttacctctct agtctgtgct agctcttcca gccccctgtc
      601 atggcatctt ccaggggtcc gagagctcag ctagtcttct tcctccaacc cgggccccta
      661 tgtccacttc aggacagcat gtttgctgcc tccagggatc ctgtgtcccc gagctgggac
      721 caccttatat tcccagggcc ggttaatgtg gctctggttc tgggtacttt tatctgtccc
      781 ctccacccca cagtgggcca agcttctgac ctcttctctt cctcccacag ggcctcgaga
      841 gatctggcag cggagagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc
      901 ccggccctag gctcgagatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
      961 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     1021 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     1081 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     1141 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     1201 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     1261 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     1321 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     1381 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     1441 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     1501 gcaagcccgg tgcctgatct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc
     1561 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     1621 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     1681 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     1741 acaatagcag gcatgctggg gatgcggtgg gctctatggg tctcgacatt gattattgac
     1801 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     1861 cgttttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc
     1921 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc
     1981 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc
     2041 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac
     2101 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc
     2161 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag
     2221 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct
     2281 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct
     2341 cgttgaccga atcaccgacc tctctcccca gggggatcca ccggagctta ccatggtgag
     2401 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     2461 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2521 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2581 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2641 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2701 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2761 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2821 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     2881 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     2941 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     3001 cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3061 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt agacgcgtga
     3121 attcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct
     3181 ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa
     3241 gccccttgag catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg
     3301 ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca
     3361 tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa
     3421 caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt
     3481 attccataga aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt
     3541 tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct
     3601 ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca
     3661 gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     3721 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     3781 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     3841 cgtgccagcg gatcgacagt actaagcttt actagggaca ggattggtga cagaaaagcc
     3901 ccatccttag gcctcctcct tcctagtctc ctgatattgg gtctaacccc cacctcctgt
     3961 taggcagatt ccttatctgg tgacacaccc ccatttcctg gagccatctc tctccttgcc
     4021 agaacctcta aggtttgctt acgatggagc cagagaggat cctgggaggg agagcttggc
     4081 agggggtggg agggaagggg gggatgcgtg acctgcccgg ttctcagtgg ccaccctgcg
     4141 ctaccctctc ccagaacctg agctgctctg acgcggctgt ctggtgcgtt tcactgatcc
     4201 tggtgctgca gcttccttac acttcccaag aggagaagca gtttggaaaa acaaaatcag
     4261 aataagttgg tcctgagttc taactttggc tcttcacctt tctagtcccc aatttatatt
     4321 gttcctccgt gcgtcagttt tacctgtgag ataaggccag tagccagccc cgtcctggca
     4381 gggctgtggt gaggaggggg gtgtccgtgt ggaaaactcc ctttgtgaga atggtgcgtc
     4441 ctaggtgttc accaggtcgt ggccgcctct actccctttc tctttctcca tccttctttc
     4501 cttaaagagt ccccagtgct atctgggaca tattcctccg cccagagcag ggtcccgctt
     4561 ccctaaggcc ctgctctggg cttctgggtt tgagtccttg gcaagcccag gagaggcgct
     4621 caggcttccc tgtccccctt cctcgtccac catctcatgc ccctggctct cctgcccctt
     4681 ccctacaggg gttcctggct ctgctctatc ccaatggcgc gccgagcttg gcgtaatcat
     4741 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4801 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4861 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4921 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4981 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5041 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5101 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5161 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5221 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5281 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5341 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5401 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5461 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5521 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5581 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5641 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5701 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5761 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5821 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5881 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5941 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6001 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6061 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6121 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6181 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6241 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6301 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6361 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6421 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6481 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6541 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6601 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6661 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6721 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6781 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6841 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     6901 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     6961 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     7021 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     7081 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
     7141 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     7201 gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     7261 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     7321 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgacgcgt attgggatcc
     7381 ctttgctt
//