LOCUS       sequence-415826-        5322 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recruited Phi29 DNA polymerase (-exo) with a C-terminal N5 coiled 
            coil (CC) domain, expressed from CMV or T7 promoters. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5322)
  AUTHORS   Ferreira da Silva J, Tou CJ, King EM, Eller ML, Rufino-Ramos D, Ma 
            L, Cromwell CR, Metovic J, Benning FMC, Chao LH, Eichler FS, 
            Kleinstiver BP
  TITLE     Click editing enables programmable genome writing using DNA 
            polymerases and HUH endonucleases.
  JOURNAL   Nat Biotechnol. 2024 Jul 22. doi: 10.1038/s41587-024-02324-x.
  PUBMED    39039307
REFERENCE   2  (bases 1 to 5322)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2024 Jul 22. doi: 10.1038/s41587-024-02324-x."
COMMENT     Sequence Label: Recruited polymerase expression -
            pCMV-T7-Phi2...
FEATURES             Location/Qualifiers
     source          1..5322
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             869..889
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1015..1032
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1055..1072)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1061..1285
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1356..1372)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1356..1372)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1369..1391)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1380..1396
                     /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(1404..1410,1411..1428,1429..1434))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1449..1470
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1587..1604)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1758..2346)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1838..1857)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2517..3308,3309..3377))
                     /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     3140..3159
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3378..3482)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3561..3580)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3752..4131
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4132..4335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4286..4306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4377..4396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4377..4395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 acggagcaga ttctacaagg gcaacgagta cctgaaaagc agcggcggcg aaatcgccga
       61 tctgtggctg agcaatgtgg acctggaact gatgaaggaa cactacgacc tgtacaacgt
      121 ggaatacatc agcggcctga agtttaaggc cacaaccggc ctgttcaaag acttcatcga
      181 taagtggacc tacatcaaaa caaccagcga gggcgctatc aagcagctgg ccaagctgat
      241 gctgaattct ctgtacggca agttcgccag caaccctgat gtcacaggca aggtgcccta
      301 cctgaaggaa aacggcgccc tgggcttcag actgggcgag gaagaaacca aggaccctgt
      361 gtacacccct atgggtgttt tcatcaccgc ctgggccaga tacaccacca tcaccgccgc
      421 tcaggcttgt tacgatagaa tcatctattg tgataccgac tctatccacc tgaccgggac
      481 cgagatcccc gacgtgatca aggatattgt ggaccccaag aaactgggct actgggccca
      541 cgagtccacc ttcaagcggg ccaagtacct gagacagaag acctacatcc aggacatcta
      601 catgaaagaa gtggacggca agctcgtgga aggcagcccc gacgactaca cagacatcaa
      661 gttcagcgtg aagtgcgccg gcatgaccga taagatcaaa aaggaggtga catttgagaa
      721 cttcaaggtg ggattcagcc gcaagatgaa acctaagcct gttcaagtgc ctggcggtgt
      781 tgtgctggtg gatgacacct tcacaatcaa gtccggcgga agctctggtg gcagcaagcg
      841 gaccgccgac ggctctgaat tcgagagccc taagaagaaa agaaaggtga gcggtgggag
      901 cagcggcggg tcaggcgaga tcgccgcact ggaagccaag atcgctgccc tgaaggcaaa
      961 gaacgccgct ctgaaggccg agatcgccgc cctggaagcc ggatactgac cggtcatcat
     1021 caccatcacc attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     1081 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     1141 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     1201 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     1261 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgata
     1321 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     1381 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag
     1441 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     1501 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     1561 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     1621 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     1681 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1741 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1801 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1861 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     1921 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     1981 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2041 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2101 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2161 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     2221 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2281 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2341 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2401 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2461 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2521 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2581 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2641 gctgcaatga taccgcgcga accacgctca ccggctccag atttatcagc aataaaccag
     2701 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     2761 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     2821 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     2881 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     2941 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3001 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3061 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3121 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3181 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3241 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     3301 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3361 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3421 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3481 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat
     3541 cccctagggt cgactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     3601 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
     3661 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
     3721 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
     3781 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     3841 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     3901 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     3961 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     4021 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     4081 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     4141 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     4201 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     4261 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     4321 tatataagca gagctggttt agtgaaccgt cagatccgct agagatccgc ggccgctaat
     4381 acgactcact atagggagag ccgccaccat gaagcacatg cctagaaaga tgtacagctg
     4441 cgacttcgag acaacaacca aggtcgagga ctgcagagtg tgggcttacg gatatatgaa
     4501 tatcgaggac cactctgagt acaagatcgg aaatagcctg gatgagttta tggcctgggt
     4561 cctgaaagtg caggccgacc tgtacttcca caacctgaaa ttcgacggcg ccttcatcat
     4621 taactggctg gaacggaacg gctttaagtg gtccgccgat ggactgccaa acacctacaa
     4681 caccatcatc agcagaatgg gccagtggta catgatcgac atctgcctgg gctacaaggg
     4741 caaaagaaag atccataccg tgatctacga cagcctgaag aaactgcctt tccccgtgaa
     4801 gaagatcgct aaagacttca agctgacagt gctgaagggc gacattgatt accacaagga
     4861 aagacctgtg ggatacaaga tcacacctga ggaatatgcc tacatcaaga acgccatcca
     4921 gatcatcgcc gaggccctgc tgatccagtt caagcagggc ctggaccgga tgaccgctgg
     4981 cagcgacagc ctcaaaggct tcaaggacat tatcaccact aagaagttca aaaaggtgtt
     5041 cccaacactg tctcttggac tggacaagga agtgcggtac gcctatagag gcggctttac
     5101 ctggctgaac gacagattca aggaaaagga aatcggcgag ggcatggtgt ttgacgtgaa
     5161 ctccctctac cctgcccaga tgtacagccg gctgctgccc tatggcgagc ctatcgtgtt
     5221 tgagggcaag tacgtgtggg atgaagatta ccccctgcac atccagcaca tccggtgcga
     5281 gttcgagctg aaggagggct acatcccaac aatccaaatc aa
//
