LOCUS       sequence-432897-        5745 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Unfused click editor construct expressing PCV2-EcKlenow, expressed 
            from CMV or T7 promoters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5745)
  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 5745)
  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: Unfused click editor construct; PCV2-EcKlenow ...
FEATURES             Location/Qualifiers
     source          1..5745
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1215..1234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1406..1785
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1786..1989
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1940..1960
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2031..2050
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2031..2049
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4385..4405
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4414..4431
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4454..4471)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4460..4684
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4755..4771)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4755..4771)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4768..4790)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4779..4795
                     /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(4803..4809,4810..4827,4828..4833))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4848..4869
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4986..5003)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5157..5745)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5237..5256)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gcgaaccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta
     1201 gggtcgactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     1261 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     1321 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     1381 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
     1441 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     1501 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     1561 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     1621 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     1681 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     1741 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     1801 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     1861 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     1921 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     1981 agcagagctg gtttagtgaa ccgtcagatc cgctagagat ccgcggccgc taatacgact
     2041 cactataggg agagccgcca ccatgagccc atctaagaaa aacggcagaa gcggacctca
     2101 acctcacaag cggtgggtct ttaccctgaa caaccccagc gaagatgaga gaaagaagat
     2161 ccgggacctg cctatcagcc tgttcgacta cttcatcgtg ggcgaggaag gaaatgagga
     2221 aggccggacc ccccacctgc agggcttcgc caacttcgtg aaaaagcaga ccttcaacaa
     2281 ggtgaagtgg tacctgggcg ctagatgcca catcgagaag gccaagggca ccgaccagca
     2341 gaacaaagag tactgcagca aggaaggcaa tctgctgatg gaatgtggcg cccctagatc
     2401 ccagggacag agatctggag gatctagcgg aggatcctct ggcagcgaga caccaggaac
     2461 aagcgagtca gcaacaccag agagcagtgg cggcagcagc ggcggcagca gcgttatctc
     2521 ctacgacaac tacgtgacca tcctggacga agagacactg aaggcttgga tcgctaaact
     2581 ggaaaaagcc cctgtgttcg ccttcgccac cgccaccgac tccttagata acatcagcgc
     2641 caacctggtg ggcctgtctt tcgccatcga gcctggcgtg gccgcctaca tccccgtggc
     2701 ccacgattac ctggatgccc cagatcaaat cagcagagag agagcactgg agctcctgaa
     2761 gcccctgctg gaggacgaga aggccctgaa ggtgggacag aacctgaaat acgatagggg
     2821 aatcctggct aattacggca tcgagctgag aggcatcgcc ttcgatacca tgctggaatc
     2881 ttacatcctg aacagcgtgg ccggccgcca cgacatggac agcctggctg aacggtggct
     2941 gaagcacaaa accatcacct tcgaagagat cgccggaaag ggcaagaacc agctgacatt
     3001 taatcagatc gcactggaag aagccggcag atacgccgct gaggacgccg acgtgaccct
     3061 tcagctgcac ctgaagatgt ggcccgatct gcagaagcac aagggcccac taaacgtgtt
     3121 cgagaacatc gagatgcctc tggtgcccgt cctgagcaga atcgagagga atggcgtgaa
     3181 gatcgacccc aaggtgctgc acaaccacag cgaggaactg acactgcggc tggccgagct
     3241 ggaaaagaag gcccacgaaa tcgccggcga ggagttcaac ctgtctagca ccaagcagct
     3301 acaaacaatc ctgttcgaga agcagggcat taagcctctg aaaaagacac ctggcggagc
     3361 ccccagcacc agcgaagaag tgctggagga actggcgctg gattaccctc tgcccaaagt
     3421 gatcctggaa tatagaggac tggccaagct gaagtctaca tacaccgaca agctgcctct
     3481 gatgatcaac cccaagaccg gccgggtgca tacatcttat caccaggccg tgacagccac
     3541 cggccggctg agcagcacag atcctaacct gcaaaacatc ccagtgcgga acgaagaggg
     3601 cagaagaatc agacaggctt tcattgcccc tgaggactac gtcatcgtgt ctgctgacta
     3661 cagccagata gaactgagaa tcatggccca tctgtctcgg gacaagggcc tgctgaccgc
     3721 ttttgcagaa ggcaaggaca tccacagagc caccgctgcc gaagtgttcg gcctgcctct
     3781 ggaaacagtg accagcgagc aaagaaggtc cgccaaggcc attaacttcg gcctgatcta
     3841 cggcatgagc gcctttggcc tggccagaca gctcaacatc cctagaaagg aggcccagaa
     3901 atacatggac ctgtacttcg agcggtaccc cggcgtgctg gagtacatgg aacggaccag
     3961 agcccaggct aaagagcagg gatacgtgga aaccctggat ggccggagac tgtatctgcc
     4021 tgacatcaag agcagcaacg gcgccagaag agccgccgct gagagagctg ctatcaacgc
     4081 ccctatgcag ggcaccgccg ccgacatcat caagcgggcc atgatcgccg tggacgcctg
     4141 gctgcaggcc gagcagccta gagtgcggat gatcatgcag gtgcacgacg agctggtgtt
     4201 tgaggtgcac aaggacgacg tggatgctgt ggccaagcag attcaccagc tgatggaaaa
     4261 ttgcaccaga ctggacgtgc cactgctggt cgaggttgga agcggagaga attgggacca
     4321 ggcccactcc ggcggaagct ctggtggcag caagcggacc gccgacggct ctgaattcga
     4381 gagccctaag aagaaaagaa aggtgtgacc ggtcatcatc accatcacca ttgagtttaa
     4441 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     4501 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     4561 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     4621 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     4681 atggcttctg aggcggaaag aaccagctgg ggctcgatac cgtcgacctc tagctagagc
     4741 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4801 cacaacatac gagccggaag cataaagtgt aaagcctagg gtgcctaatg agtgagctaa
     4861 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4921 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4981 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5041 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5101 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5161 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5221 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5281 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5341 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5401 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5461 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5521 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5581 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5641 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5701 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaa
//
