LOCUS       sequence-415830-        5322 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Recruited ePhi29 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 Apr 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-ePhi...
FEATURES             Location/Qualifiers
     source          1..5322
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2191..2211
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2337..2354
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2377..2394)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2383..2607
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2678..2694)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2678..2694)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2691..2713)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2702..2718
                     /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(2726..2732,2733..2750,2751..2756))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2771..2792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2909..2926)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3080..3668)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3160..3179)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3839..4630,4631..4699))
                     /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     4462..4481
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4700..4804)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4883..4902)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(5074..5322,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaagcacatg
      421 cctagaaaga gatacagctg cgacttcgag acaacaacca aggtcgagga ctgcagagtg
      481 tgggcttacg gatatatgaa tatcgaggac cactctgagt acaagatcgg aaatagcctg
      541 gatgagttta tggcctgggc cctgaaagtg caggccgacc tgtacttcca caacctgaaa
      601 ttcgacggcg ccttcatcat taactggctg gaacggaacg gctttaagtg gtccgccgat
      661 ggactgccaa acacctacaa caccatcatc agcagaaccg gccagtggta catgatcgac
      721 atctgcctgg gctacaaggg caaaagaaag atccataccg tgatctacga cagcctgaag
      781 aaactgcctt tccccgtgaa gaagatcgct aaagacttca agctgacagt gctgaagggc
      841 gacattgatt accacaagga aagacctgtg ggatacaaga tcacacctga ggaatatgcc
      901 tacatcaaga acgccatcca gatcatcgcc gaggccctgc tgatccagtt caagcagggc
      961 ctggaccgga tgaccgctgg cagcgacagc ctcaaagact tcaaggacat tatcaccact
     1021 aagaagttca aaaaggtgtt cccaacactg tctcttggac tggacaagaa ggtgcggtac
     1081 gcctatagag gcggctttac ctggctgaac gacagattca aggaaaagga aatcggcgag
     1141 ggcatggtgt ttgacgtgaa ctccctctac cctgcccaga tgtacagccg gctgctgccc
     1201 tatggcgagc ctatcgtgtt tgagggcaag tacgtgtggg atgaagatta ccccctgcac
     1261 atccagcaca tccggtgcga gttcgagctg aaggagggct acatcccaac aatccaaatc
     1321 aaacggagca gattctacaa gggcaacgag tacctgaaaa gcagcggcgg cgaaatcgcc
     1381 gatctgtggc tgagcaatgt ggacctggaa ctgatgaagg aacactacga cctgtacaac
     1441 gtggaataca tcagcggcct gaagtttaag gccacaaccg gcctgttcaa agacttcatc
     1501 gataagtgga cctacatcaa aacaaccagc gagggcgcta tcaagcagct ggccaagctg
     1561 atgctgaatt ctctgtacgg caagttcgcc agcaaccctg atgtcacagg caaggtgccc
     1621 tacctgaagg aaaacggcgc cctgggcttc agactgggcg aggaagaaac caaggaccct
     1681 gtgtacaccc ctatgggtgt tttcatcacc gcctgggcca gatacaccac catcaccgcc
     1741 gctcaggctt gttacgatag aatcatctat tgtgataccg actctatcca cctgaccggg
     1801 accgagatcc ccgacgtgat caaggatatt gtggacccca agaaactggg ctactgggcc
     1861 cacgagtcca ccttcaagcg ggccaagtac ctgagaccca agacctacat ccaggacatc
     1921 tacatgaaag aagtggacgg cgagctcgtg gaaggcagcc ccgacgacta cacagacatc
     1981 aagctgagcg tgaagtgcgc cggcatgacc gataagatca aaaaggaggt gacatttgag
     2041 aacttcaagg tgggattcag ccgcaagatg aaacctaagc ctgttcaagt gcctggcggt
     2101 gttgtgctgg tggatgacac cttcacaatc aagtccggcg gaagctctgg tggcagcaag
     2161 cggaccgccg acggctctga attcgagagc cctaagaaga aaagaaaggt gagcggtggg
     2221 agcagcggcg ggtcaggcga gatcgccgca ctggaagcca agatcgctgc cctgaaggca
     2281 aagaacgccg ctctgaaggc cgagatcgcc gccctggaag ccggatactg accggtcatc
     2341 atcaccatca ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     2401 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2461 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2521 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     2581 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga
     2641 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2701 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2761 agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2821 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2881 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2941 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3001 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3061 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3121 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3181 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3241 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3301 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3361 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3421 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3481 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3541 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3601 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3661 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3721 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3781 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3841 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3901 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3961 gtgctgcaat gataccgcgc gaaccacgct caccggctcc agatttatca gcaataaacc
     4021 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4081 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4141 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4201 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4261 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4321 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4381 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4441 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4501 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4561 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4621 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4681 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4741 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4801 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg
     4861 atcccctagg gtcgactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     4921 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     4981 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     5041 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
     5101 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     5161 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     5221 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     5281 atttacggta aactgcccac ttggcagtac atcaagtgta tc
//
