LOCUS       sequence-432904-        5775 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Unfused click editor construct expressing mSA-EcKlenow, expressed 
            from CMV or T7 promoters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5775)
  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 5775)
  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; mSA-EcKlenow f...
FEATURES             Location/Qualifiers
     source          1..5775
                     /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"
     regulatory      2057..2066
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2090..2431
                     /codon_start=1
                     /product="monomeric streptavidin/rhizavidin hybrid (Lim et 
                     al., 2013)"
                     /label=mSA
                     /translation="AEAGITGTWYNQSGSTFTVTAGADGNLTGQYENRAQGTGCQNSPY
                     TLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATE
                     QGQDTFTKVK"
     CDS             2507..2527
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4415..4435
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4444..4461
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4484..4501)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4490..4714
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4785..4801)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4785..4801)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4798..4820)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4809..4825
                     /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(4833..4839,4840..4857,4858..4863))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4878..4899
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5016..5033)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5187..5775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5267..5286)
                     /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 ccatgggggc ccagccggcc agatctatgg cggaagcggg
     2101 tatcaccggc acgtggtaca accagtctgg ttctaccttc accgttaccg cgggtgcgga
     2161 cggtaacctg accggtcagt acgaaaaccg tgcgcagggc actggttgcc agaactctcc
     2221 gtacaccctg accggtcgtt acaacggtac caaactggaa tggcgtgttg aatggaacaa
     2281 ctctaccgaa aactgccact ctcgtaccga atggcgtggt cagtaccagg gtggtgcgga
     2341 agcgcgtatc aacacccagt ggaacctgac ctacgaaggt ggttctggtc cggcgaccga
     2401 acagggtcag gacaccttca ccaaagttaa accgtctgcg gcgtctggat ccagcgctgg
     2461 aggcggtggc tccggaggag gcggaagcgg aggaggaggt agcggaccta agaaaaagag
     2521 gaaggtggcg gccgctggat ccgttatctc ctacgacaac tacgtgacca tcctggacga
     2581 agagacactg aaggcttgga tcgctaaact ggaaaaagcc cctgtgttcg ccttcgccac
     2641 cgccaccgac tccttagata acatcagcgc caacctggtg ggcctgtctt tcgccatcga
     2701 gcctggcgtg gccgcctaca tccccgtggc ccacgattac ctggatgccc cagatcaaat
     2761 cagcagagag agagcactgg agctcctgaa gcccctgctg gaggacgaga aggccctgaa
     2821 ggtgggacag aacctgaaat acgatagggg aatcctggct aattacggca tcgagctgag
     2881 aggcatcgcc ttcgatacca tgctggaatc ttacatcctg aacagcgtgg ccggccgcca
     2941 cgacatggac agcctggctg aacggtggct gaagcacaaa accatcacct tcgaagagat
     3001 cgccggaaag ggcaagaacc agctgacatt taatcagatc gcactggaag aagccggcag
     3061 atacgccgct gaggacgccg acgtgaccct tcagctgcac ctgaagatgt ggcccgatct
     3121 gcagaagcac aagggcccac taaacgtgtt cgagaacatc gagatgcctc tggtgcccgt
     3181 cctgagcaga atcgagagga atggcgtgaa gatcgacccc aaggtgctgc acaaccacag
     3241 cgaggaactg acactgcggc tggccgagct ggaaaagaag gcccacgaaa tcgccggcga
     3301 ggagttcaac ctgtctagca ccaagcagct acaaacaatc ctgttcgaga agcagggcat
     3361 taagcctctg aaaaagacac ctggcggagc ccccagcacc agcgaagaag tgctggagga
     3421 actggcgctg gattaccctc tgcccaaagt gatcctggaa tatagaggac tggccaagct
     3481 gaagtctaca tacaccgaca agctgcctct gatgatcaac cccaagaccg gccgggtgca
     3541 tacatcttat caccaggccg tgacagccac cggccggctg agcagcacag atcctaacct
     3601 gcaaaacatc ccagtgcgga acgaagaggg cagaagaatc agacaggctt tcattgcccc
     3661 tgaggactac gtcatcgtgt ctgctgacta cagccagata gaactgagaa tcatggccca
     3721 tctgtctcgg gacaagggcc tgctgaccgc ttttgcagaa ggcaaggaca tccacagagc
     3781 caccgctgcc gaagtgttcg gcctgcctct ggaaacagtg accagcgagc aaagaaggtc
     3841 cgccaaggcc attaacttcg gcctgatcta cggcatgagc gcctttggcc tggccagaca
     3901 gctcaacatc cctagaaagg aggcccagaa atacatggac ctgtacttcg agcggtaccc
     3961 cggcgtgctg gagtacatgg aacggaccag agcccaggct aaagagcagg gatacgtgga
     4021 aaccctggat ggccggagac tgtatctgcc tgacatcaag agcagcaacg gcgccagaag
     4081 agccgccgct gagagagctg ctatcaacgc ccctatgcag ggcaccgccg ccgacatcat
     4141 caagcgggcc atgatcgccg tggacgcctg gctgcaggcc gagcagccta gagtgcggat
     4201 gatcatgcag gtgcacgacg agctggtgtt tgaggtgcac aaggacgacg tggatgctgt
     4261 ggccaagcag attcaccagc tgatggaaaa ttgcaccaga ctggacgtgc cactgctggt
     4321 cgaggttgga agcggagaga attgggacca ggcccactcc ggcggaagct ctggtggcag
     4381 caagcggacc gccgacggct ctgaattcga gagccctaag aagaaaagaa aggtgtgacc
     4441 ggtcatcatc accatcacca ttgagtttaa acccgctgat cagcctcgac tgtgccttct
     4501 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     4561 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     4621 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     4681 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     4741 ggctcgatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4801 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4861 aaagcctagg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4921 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4981 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5041 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5101 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5161 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5221 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5281 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5341 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5401 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5461 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5521 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5581 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5641 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5701 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5761 aaaaaaggat ctcaa
//
