LOCUS       sequence-415816-        5415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recruited EcKlenow 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 5415)
  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 5415)
  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-EcKl...
FEATURES             Location/Qualifiers
     source          1..5415
                     /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"
     regulatory      403..412
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2284..2304
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2430..2447
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2470..2487)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2476..2700
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2771..2787)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2771..2787)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2784..2806)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2795..2811
                     /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(2819..2825,2826..2843,2844..2849))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2864..2885
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3002..3019)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3173..3761)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3253..3272)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3932..4723,4724..4792))
                     /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     4555..4574
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4793..4897)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4976..4995)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(5167..5415,1..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 ggttatctcc
      421 tacgacaact acgtgaccat cctggacgaa gagacactga aggcttggat cgctaaactg
      481 gaaaaagccc ctgtgttcgc cttcgccacc gccaccgact ccttagataa catcagcgcc
      541 aacctggtgg gcctgtcttt cgccatcgag cctggcgtgg ccgcctacat ccccgtggcc
      601 cacgattacc tggatgcccc agatcaaatc agcagagaga gagcactgga gctcctgaag
      661 cccctgctgg aggacgagaa ggccctgaag gtgggacaga acctgaaata cgatagggga
      721 atcctggcta attacggcat cgagctgaga ggcatcgcct tcgataccat gctggaatct
      781 tacatcctga acagcgtggc cggccgccac gacatggaca gcctggctga acggtggctg
      841 aagcacaaaa ccatcacctt cgaagagatc gccggaaagg gcaagaacca gctgacattt
      901 aatcagatcg cactggaaga agccggcaga tacgccgctg aggacgccga cgtgaccctt
      961 cagctgcacc tgaagatgtg gcccgatctg cagaagcaca agggcccact aaacgtgttc
     1021 gagaacatcg agatgcctct ggtgcccgtc ctgagcagaa tcgagaggaa tggcgtgaag
     1081 atcgacccca aggtgctgca caaccacagc gaggaactga cactgcggct ggccgagctg
     1141 gaaaagaagg cccacgaaat cgccggcgag gagttcaacc tgtctagcac caagcagcta
     1201 caaacaatcc tgttcgagaa gcagggcatt aagcctctga aaaagacacc tggcggagcc
     1261 cccagcacca gcgaagaagt gctggaggaa ctggcgctgg attaccctct gcccaaagtg
     1321 atcctggaat atagaggact ggccaagctg aagtctacat acaccgacaa gctgcctctg
     1381 atgatcaacc ccaagaccgg ccgggtgcat acatcttatc accaggccgt gacagccacc
     1441 ggccggctga gcagcacaga tcctaacctg caaaacatcc cagtgcggaa cgaagagggc
     1501 agaagaatca gacaggcttt cattgcccct gaggactacg tcatcgtgtc tgctgactac
     1561 agccagatag aactgagaat catggcccat ctgtctcggg acaagggcct gctgaccgct
     1621 tttgcagaag gcaaggacat ccacagagcc accgctgccg aagtgttcgg cctgcctctg
     1681 gaaacagtga ccagcgagca aagaaggtcc gccaaggcca ttaacttcgg cctgatctac
     1741 ggcatgagcg cctttggcct ggccagacag ctcaacatcc ctagaaagga ggcccagaaa
     1801 tacatggacc tgtacttcga gcggtacccc ggcgtgctgg agtacatgga acggaccaga
     1861 gcccaggcta aagagcaggg atacgtggaa accctggatg gccggagact gtatctgcct
     1921 gacatcaaga gcagcaacgg cgccagaaga gccgccgctg agagagctgc tatcaacgcc
     1981 cctatgcagg gcaccgccgc cgacatcatc aagcgggcca tgatcgccgt ggacgcctgg
     2041 ctgcaggccg agcagcctag agtgcggatg atcatgcagg tgcacgacga gctggtgttt
     2101 gaggtgcaca aggacgacgt ggatgctgtg gccaagcaga ttcaccagct gatggaaaat
     2161 tgcaccagac tggacgtgcc actgctggtc gaggttggaa gcggagagaa ttgggaccag
     2221 gcccactccg gcggaagctc tggtggcagc aagcggaccg ccgacggctc tgaattcgag
     2281 agccctaaga agaaaagaaa ggtgagcggt gggagcagcg gcgggtcagg cgagatcgcc
     2341 gcactggaag ccaagatcgc tgccctgaag gcaaagaacg ccgctctgaa ggccgagatc
     2401 gccgccctgg aagccggata ctgaccggtc atcatcacca tcaccattga gtttaaaccc
     2461 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     2521 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     2581 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2641 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     2701 cttctgaggc ggaaagaacc agctggggct cgataccgtc gacctctagc tagagcttgg
     2761 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     2821 acatacgagc cggaagcata aagtgtaaag cctagggtgc ctaatgagtg agctaactca
     2881 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     2941 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     3001 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3061 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3121 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3181 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3241 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3301 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3361 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3421 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3481 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3541 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3601 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3661 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3721 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3781 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3841 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3901 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3961 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4021 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgcgaaccac
     4081 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4141 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4201 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4261 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4321 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4381 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4441 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4501 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4561 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4621 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4681 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4741 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4801 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4861 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4921 ctgacgtcga cggatcggga gatcgatctc ccgatcccct agggtcgact ctcagtacaa
     4981 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     5041 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     5101 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
     5161 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     5221 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     5281 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     5341 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     5401 tacatcaagt gtatc
//
