LOCUS       sequence-415814-        5205 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Unfused ePhi29 DNA polymerase (-exo), expressed from CMV or T7 
            promoters. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5205)
  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 5205)
  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: Polymerase expression - pCMV-ePhi29 (LM2802)
FEATURES             Location/Qualifiers
     source          1..5205
                     /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             2220..2237
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2260..2277)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2266..2490
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2561..2577)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2561..2577)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2574..2596)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2585..2601
                     /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(2609..2615,2616..2633,2634..2639))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2654..2675
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2792..2809)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2963..3551)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3043..3062)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3722..4513,4514..4582))
                     /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     4345..4364
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4583..4687)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4766..4785)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(4957..5205,1..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 gtgaccggtc
     2221 atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     2281 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2341 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2401 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2461 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2521 cgataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     2581 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     2641 cctagggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     2701 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     2761 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     2821 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     2881 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     2941 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3001 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3061 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3121 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3181 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3241 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3301 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3361 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     3421 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3481 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3541 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3601 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     3661 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     3721 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     3781 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     3841 ccagtgctgc aatgataccg cgcgaaccac gctcaccggc tccagattta tcagcaataa
     3901 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     3961 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4021 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4081 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4141 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4201 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4261 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4321 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4381 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4441 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4501 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     4561 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     4621 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     4681 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatcgatctc
     4741 ccgatcccct agggtcgact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4801 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     4861 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
     4921 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
     4981 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     5041 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     5101 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     5161 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatc
//
