LOCUS       IVT-MCP-Phi29       5442 bp    DNA     circular SYN 14-APR-2025
DEFINITION  in vitro transcription of mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5442)
  AUTHORS   Liu B, Dong X, Zheng C, Keener D, Chen Z, Cheng H, Watts JK, Xue W, 
            Sontheimer EJ
  TITLE     Targeted genome editing with a DNA-dependent DNA polymerase and 
            exogenous DNA-containing templates.
  JOURNAL   Nat Biotechnol. 2023 Sep 14. doi: 10.1038/s41587-023-01947-w.
  PUBMED    37709915
REFERENCE   2  (bases 1 to 5442)
  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: "Nat 
            Biotechnol. 2023 Sep 14. doi: 10.1038/s41587-023-01947-w."
COMMENT     Sequence Label: IVT-MCP-Phi29
FEATURES             Location/Qualifiers
     source          1..5442
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      109..118
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             118..504
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             2326..2346
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2752..2769)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2758..2982
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3053..3069)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3053..3069)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3066..3088)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3077..3093
                     /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(3101..3107,3108..3125,3126..3131))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3146..3167
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3284..3301)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3455..4043)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3535..3554)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4214..5005,5006..5074))
                     /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     4837..4856
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5075..5179)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5258..5277)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggtttagtga accgtcagat ccgctagaga tccgcggccg ctaatacgac tcactataag
       61 gagaataaac tagtattctt ctggtcccca cagactcaga gagaacccgc caccatggct
      121 tcaaacttta ctcagttcgt gctcgtggac aatggtggga caggggatgt gacagtggct
      181 ccttctaatt tcgctaatgg ggtggcagag tggatcagct ccaactcacg gagccaggcc
      241 tacaaggtga catgcagcgt caggcagtct agtgcccaga agagaaagta taccatcaag
      301 gtggaggtcc ccaaagtggc tacccagaca gtgggcggag tcgaactgcc tgtcgccgct
      361 tggaggtcct acctgaacat ggagctcact atcccaattt tcgctaccaa ttctgactgt
      421 gaactcatcg tgaaggcaat gcaggggctc ctcaaagacg gtaatcctat cccttccgcc
      481 atcgccgcta actcaggtat ctactctgga ggatctagcg gaggatcctc tggcagcgag
      541 acaccaggaa caagcgagtc agcaacacca gagagcagtg gcggcagcag cggcggcagc
      601 agcaagcata tgcccagaaa aatgtattcc tgtgattttg agaccacaac caaagtagaa
      661 gattgtcggg tttgggccta cggatacatg aacattgaag accattcaga atataaaatc
      721 ggtaacagtc tcgatgaatt catggcatgg gtgttgaaag tccaagccga tctttatttc
      781 cataacttga aatttgacgg cgcttttatc attaactggc ttgagcgcaa tggcttcaag
      841 tggtcagcgg atgggcttcc aaacacgtat aatacgatta tatcccgcat gggtcagtgg
      901 tacatgatcg acatatgtct tggttacaaa ggcaaacgca agatccatac cgttatctat
      961 gacagtttga agaagttgcc gtttccagtt aagaaaattg cgaaagactt taaacttacg
     1021 gtgcttaagg gcgacatcga ctaccataag gaacggccag ttggttacaa gataacgcct
     1081 gaggagtacg cttatatcaa gaatgacata cagattatag ctgaagcatt gctcatacag
     1141 ttcaaacaag gcctggatag gatgaccgct ggcagtgatt cacttaaagg atttaaagac
     1201 ataattacta cgaagaagtt taagaaagtg tttccaaccc tcagtctggg cctcgacaaa
     1261 gaagtgcgat atgcgtaccg cggcgggttc acatggctta atgaccgatt caaggaaaag
     1321 gaaataggag aagggatggt tttcgatgtt aatagcctct atcccgcgca aatgtacagt
     1381 aggcttttgc catatggcga accaatcgtg ttcgagggga agtatgtatg ggatgaggac
     1441 tatcctctcc acattcagca catccggtgt gagttcgagc ttaaagaggg atatatccct
     1501 actatccaga taaaacgctc tcgattttac aaggggaacg aataccttaa gtcatccggg
     1561 ggggaaatcg cagatttgtg gttgtcaaat gttgaccttg agttgatgaa ggaacactat
     1621 gatctctaca acgtcgaata catttcaggt ttgaaattca aggcaaccac cgggcttttt
     1681 aaagatttca tcgataaatg gacctacata aaaacgacct cagaaggagc cataaagcag
     1741 ttggctaaac ttatgcttaa ttctctctac ggaaaatttg caagtaatcc tgacgttaca
     1801 gggaaagtcc cgtacttgaa agaaaatgga gcgctggggt ttaggctcgg tgaggaagaa
     1861 accaaggatc cagtctacac accaatgggc gtatttatca ctgcatgggc gcgatataca
     1921 acaatcacgg ccgctcaggc ctgttacgat cgcataattt actgcgacac cgattcaatc
     1981 caccttaccg gaacagagat cccggacgtt attaaagata ttgtggaccc aaaaaagctg
     2041 ggctactggg cccacgagtc caccttcaag cgcgccaagt atctgcggca aaaaacttac
     2101 atccaggata tatacatgaa ggaggtagac ggtaagcttg tcgaaggctc accggatgac
     2161 tataccgata tcaaattctc agtaaagtgt gctggtatga ctgacaagat aaagaaggaa
     2221 gttaccttcg agaacttcaa ggtagggttc agtagaaaaa tgaaacccaa gccagtgcaa
     2281 gtgcctggtg gagtagtact ggtggacgat acattcacga tcaaacccaa gaagaagagg
     2341 aaagtctaat gactcgagct ggtactgcat gcacgcaatg ctagctgccc ctttcccgtc
     2401 ctgggtaccc cgagtctccc ccgacctcgg gtcccaggta tgctcccacc tccacctgcc
     2461 ccactcacca cctctgctag ttccagacac ctcccaagca cgcagcaatg cagctcaaaa
     2521 cgcttagcct agccacaccc ccacgggaaa cagcagtgat taacctttag caataaacga
     2581 aagtttaact aagctatact aaccccaggg ttggtcaatt tcgtgccagc cacaccctgg
     2641 agctagcaaa aaaaaaaaaa aaaaaaaaaa aaaagcatat gactaaaaaa aaaaaaaaaa
     2701 aaaaaaaaaa aaaaaaaaaa aaaaaagaga cggtttaaac ccgctgatca gcctcgactg
     2761 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2821 aaggtgccac tcccactgtc ctttcctaat aaaatgagaa aattgcatcg cattgtctga
     2881 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2941 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3001 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     3061 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3121 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3181 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3241 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     3301 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3361 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3421 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3481 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3541 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3601 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3661 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3721 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3781 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3841 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     3901 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3961 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4021 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacactc
     4081 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4141 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4201 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4261 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4321 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4381 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4441 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4501 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4561 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4621 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4681 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4741 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4801 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     4861 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     4921 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     4981 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5041 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5101 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5161 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     5221 gagatcgatc tcccgatccc ctagggtcta ctctcagtac aatctgctct gatgccgcat
     5281 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     5341 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     5401 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata ta
//