LOCUS       EK0208 CMV-TO-BFPmut-stop-t70587 (FLP-IN)                6022 bp ds-DNA     circular SYN 25-OCT-2022
DEFINITION  Expression of Trigger 1 in 3' UTR of an inactive BFP in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    EK0208 CMV-TO-BFPmut-stop-t70587 (FLP-IN)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6022)
  AUTHORS   Kaseniit E, Gao X
  TITLE     RADAR plasmids
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6022)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 25, 2022 from SnapGene Server 1.1.58
            //gt12365.cn
FEATURES             Location/Qualifiers
     source          1..6022
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        384..587
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     538..558
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    589..607
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    610..628
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     638..662
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      754..763
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             769..1476
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHAVAVA
                     RYCDLPSKLGHKLN"
     CDS             1495..1521
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1624..1653
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1709..1726)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1715..1939
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2111..2130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2262..2309
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2317..3339
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3469..3590
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3506..3525)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3560..3579
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3639..3655)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3639..3655)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3652..3674)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3663..3679
                     /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(3687..3717)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3732..3753
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3870..3887)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4041..4629)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4121..4140)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4800..5660)
                     /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     5423..5442
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5661..5765)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5840..5859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      421 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      481 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      541 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg
      601 atagagatct ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga
      661 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      721 gcctccggac tctagcgttt aaacttaagc ttcgccacca tgggatccgt gtctaagggc
      781 gaagagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac cgtggacaac
      841 catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg
      901 agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct ggctactagc
      961 ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga cttcttcaag
     1021 cagtccttcc ctgagggctt cacatgggag agagtcacca catacgagga cgggggcgtg
     1081 ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaagatc
     1141 agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact cggctgggag
     1201 gccttcaccg aaacgctgta ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc
     1261 ctgaagctcg tgggcgggag ccatctgatc gcaaacgcca agaccacata tagatccaag
     1321 aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag actggaaaga
     1381 atcaaggagg ccaacaacga aacctacgtc gagcagcacg cggtggcagt ggccagatac
     1441 tgcgacctcc ctagcaaact ggggcacaaa cttaattgaa gctagctggc tggataccca
     1501 tacgatgttc cagattacgc tctcgagata gctctctatt tgcaccagtg gtctatcggt
     1561 acgtaaggcc caacaatgac gcggatagaa ggaaacactc atccgtgtcg acttggtggg
     1621 cccgaacaaa aactcatctc agaagaggat ctgaatagcg ccgtcgacca tcatcataat
     1681 catcattgag tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     1741 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1801 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     1861 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     1921 gatgcggtgg gctctatgga gatctatcga tgttctgggg atgcggtggg ctctatggct
     1981 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2041 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2101 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2161 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2221 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt
     2281 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg
     2341 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     2401 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     2461 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     2521 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     2581 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     2641 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     2701 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     2761 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     2821 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     2881 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     2941 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     3001 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     3061 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     3121 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     3181 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     3241 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     3301 aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat
     3361 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3421 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     3481 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3541 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     3601 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     3661 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     3721 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     3781 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3841 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3901 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3961 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4021 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4081 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4141 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4201 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4261 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4321 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4381 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4441 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4501 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4561 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4621 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4681 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4741 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4801 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4861 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4921 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4981 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5041 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5101 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5161 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5221 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5281 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5341 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5401 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5461 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5521 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5581 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5641 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5701 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5761 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     5821 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     5881 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     5941 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     6001 ttcgtacggg ccagatatac gc
//