LOCUS       sequence-432878-        8967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Variant CE1 construct with SaCas9, expressed from CMV or T7 
            promoters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8967)
  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 8967)
  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: SaCas9 variant click editor (CE1) -
            pCMV-T7-PC...
FEATURES             Location/Qualifiers
     source          1..8967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(113..217)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(296..315)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        487..866
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        867..1070
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1021..1041
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1112..1131
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1112..1130
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4783..4803
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6688..6708
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6717..6734
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6757..6774)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6763..6987
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(7058..7074)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7058..7074)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7071..7093)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7082..7098
                     /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(7106..7112,7113..7130,7131..7136))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7151..7172
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7289..7306)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7460..8048)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7540..7559)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8219..8967,1..43,44..112))
                     /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     8842..8861
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
       61 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
      121 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
      181 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
      241 ctgacgtcga cggatcggga gatcgatctc ccgatcccct agggtcgact ctcagtacaa
      301 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      361 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      421 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      481 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      541 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      601 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      661 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      721 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      781 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      841 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      901 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      961 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     1021 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga
     1081 accgtcagat ccgctagaga tccgcggccg ctaatacgac tcactatagg gagagccgcc
     1141 accatgagcc catctaagaa aaacggcaga agcggacctc aacctcacaa gcggtgggtc
     1201 tttaccctga acaaccccag cgaagatgag agaaagaaga tccgggacct gcctatcagc
     1261 ctgttcgact acttcatcgt gggcgaggaa ggaaatgagg aaggccggac cccccacctg
     1321 cagggcttcg ccaacttcgt gaaaaagcag accttcaaca aggtgaagtg gtacctgggc
     1381 gctagatgcc acatcgagaa ggccaagggc accgaccagc agaacaaaga gtactgcagc
     1441 aaggaaggca atctgctgat ggaatgtggc gcccctagat cccagggaca gagaagcgga
     1501 ggctctagcg gctccgagac acccggcaca tctgagtccg ccacccctga gagcagcggc
     1561 ggcagcggga aacggaacta catcctgggg cttgacattg ggataaccag cgttggctac
     1621 ggaattattg attatgagac acgcgatgtg attgacgccg gggttaggct gttcaaagag
     1681 gccaacgttg aaaacaacga gggaagacgg agtaagcgcg gagcaagaag actcaagcgc
     1741 agacggagac atcggattca gagggtgaaa aagctgctct tcgattacaa tctcctgacc
     1801 gatcatagtg agctgagcgg aatcaacccc tacgaggcgc gagtgaaagg gctttcccag
     1861 aagctgtccg aagaggagtt ctccgccgcg ttgctgcacc tggccaaacg gaggggggtt
     1921 cacaatgtaa acgaagtgga ggaggacacg ggcaatgaac ttagtacgaa agaacagatc
     1981 agtaggaact ctaaggctct cgaagagaaa tacgtcgctg agttgcagct tgagagactg
     2041 aaaaaagacg gcgaagtacg cggatctatt aataggttca agacttcaga ttacgtaaag
     2101 gaagccaagc agctcctgaa agtacagaaa gcgtaccatc agctcgatca gagcttcatc
     2161 gatacctaca tagatttgct ggagacacgg aggacatact acgagggccc aggggaagga
     2221 tctccttttg ggtggaagga catcaaggaa tggtacgaga tgcttatggg acattgtaca
     2281 tattttccgg aggagctcag gagcgtcaag tacgcctaca atgccgacct gtacaatgcc
     2341 ctcaatgacc tcaataacct cgtgattacc agggacgaga acgagaagct ggagtactat
     2401 gaaaagttcc agattatcga gaatgtgttt aagcagaaga agaagccgac acttaagcag
     2461 attgcaaagg aaatcctcgt gaatgaggaa gatatcaagg gatacagagt gacaagtaca
     2521 ggcaagcccg agttcacaaa tctgaaggtg taccacgata ttaaggacat aaccgcacga
     2581 aaggagataa tcgaaaacgc tgagctcctc gatcagatcg caaaaattct taccatctac
     2641 cagtctagtg aggacattca ggaggaactg actaatctga acagtgagct cacccaagag
     2701 gaaattgagc agatttcaaa cctgaaaggc tacaccggga cgcacaatct gagcctcaaa
     2761 gcaatcaacc tcattctgga tgaactttgg cacacaaatg acaaccaaat tgccatattc
     2821 aaccgcctga aactggtgcc aaaaaaagtg gatctgtcac agcaaaagga aatccctaca
     2881 accttggttg acgattttat tctgtccccc gttgtcaagc ggagcttcat ccagtcaatc
     2941 aaggtgatca atgccatcat taaaaaatac ggattgccaa acgatataat tatcgagctt
     3001 gcacgagaga agaactcaaa ggacgcccag aagatgatta acgaaatgca gaagcgcaac
     3061 cgccagacaa acgaacgcat agaggaaatt ataagaacaa ccggcaaaga gaatgccaag
     3121 tatctgatcg agaaaatcaa gctgcacgac atgcaagaag gcaagtgcct gtactctctg
     3181 gaagctatcc cactcgaaga tctgctgaat aatccattca attacgaggt ggaccacatc
     3241 atccctagat ccgtaagctt tgacaattcc ttcaataaca aagttctggt taaacaggag
     3301 gaagcctcta aaaaagggaa ccggaccccg ttccagtacc tgagctccag tgacagcaag
     3361 attagctacg agacttttaa gaaacatatt ctgaatctgg ccaaaggcaa aggcaggatc
     3421 agcaagacca agaaggagta cctcctcgaa gaacgcgaca ttaacagatt tagtgtgcag
     3481 aaagatttca tcaaccgaaa ccttgtcgat actcggtacg ccacgagagg cctgatgaat
     3541 ctcctcagga gctacttccg cgtcaataat ctggacgtta aagtcaagag cataaatggg
     3601 ggattcacca gctttctgag gagaaagtgg aagtttaaga aggaacgaaa caaaggatac
     3661 aagcaccatg ctgaggatgc tttgatcatc gctaacgcgg actttatctt taaggaatgg
     3721 aaaaagctgg ataaggcaaa gaaagtgatg gaaaaccaga tgttcgagga gaagcaggca
     3781 gagtcaatgc ctgagatcga gacagagcag gaatacaagg aaattttcat cacccctcat
     3841 cagattaaac acataaagga cttcaaagac tataaatact ctcatagggt ggacaaaaaa
     3901 cccaatcgcg agctcattaa tgacaccctg tactcaacac ggaaggatga taaaggtaat
     3961 accttgattg tgaataatct taatggattg tatgacaaag ataacgacaa gctcaagaag
     4021 ctgatcaaca agtctccaga gaagctcctt atgtatcacc acgacccaca gacttatcag
     4081 aaattgaaac tgatcatgga gcaatacggg gatgagaaga acccactcta caaatattat
     4141 gaggaaacag gtaattacct gaccaagtac tccaagaagg ataacggacc agtgatcaaa
     4201 aagataaagt actatggcaa caaacttaat gcgcatttgg acataactga cgattacccc
     4261 aattctcgaa acaaggttgt gaagctctcc ctgaagcctt atagatttga cgtgtacctg
     4321 gataatgggg tttataaatt cgtcaccgtg aaaaatctgg acgtgatcaa aaaggagaac
     4381 tattatgaag taaactcaaa gtgctatgag gaggcgaaga agctgaagaa gatctccaat
     4441 caggccgagt tcatcgcttc cttctataat aacgatctca tcaagatcaa tggagagctt
     4501 tatcgcgtca ttggtgtgaa caatgacttg ctgaacagga tcgaagtcaa tatgatagac
     4561 attacctacc gggagtatct cgaaaacatg aatgataaac ggccgcctcg catcatcaag
     4621 acaatcgcat ctaaaactca gtcaataaaa aagtactcta ccgatatcct ggggaatctc
     4681 tatgaagtga agtcaaagaa gcacccacaa atcattaaaa aaggttccgg cggaagctct
     4741 ggtggcagca agcggaccgc cgacggctct gaattcgaga gccctaagaa gaaaagaaag
     4801 gtgagcggag gctctagcgg cggaagcgtt atctcctacg acaactacgt gaccatcctg
     4861 gacgaagaga cactgaaggc ttggatcgct aaactggaaa aagcccctgt gttcgccttc
     4921 gccaccgcca ccgactcctt agataacatc agcgccaacc tggtgggcct gtctttcgcc
     4981 atcgagcctg gcgtggccgc ctacatcccc gtggcccacg attacctgga tgccccagat
     5041 caaatcagca gagagagagc actggagctc ctgaagcccc tgctggagga cgagaaggcc
     5101 ctgaaggtgg gacagaacct gaaatacgat aggggaatcc tggctaatta cggcatcgag
     5161 ctgagaggca tcgccttcga taccatgctg gaatcttaca tcctgaacag cgtggccggc
     5221 cgccacgaca tggacagcct ggctgaacgg tggctgaagc acaaaaccat caccttcgaa
     5281 gagatcgccg gaaagggcaa gaaccagctg acatttaatc agatcgcact ggaagaagcc
     5341 ggcagatacg ccgctgagga cgccgacgtg acccttcagc tgcacctgaa gatgtggccc
     5401 gatctgcaga agcacaaggg cccactaaac gtgttcgaga acatcgagat gcctctggtg
     5461 cccgtcctga gcagaatcga gaggaatggc gtgaagatcg accccaaggt gctgcacaac
     5521 cacagcgagg aactgacact gcggctggcc gagctggaaa agaaggccca cgaaatcgcc
     5581 ggcgaggagt tcaacctgtc tagcaccaag cagctacaaa caatcctgtt cgagaagcag
     5641 ggcattaagc ctctgaaaaa gacacctggc ggagccccca gcaccagcga agaagtgctg
     5701 gaggaactgg cgctggatta ccctctgccc aaagtgatcc tggaatatag aggactggcc
     5761 aagctgaagt ctacatacac cgacaagctg cctctgatga tcaaccccaa gaccggccgg
     5821 gtgcatacat cttatcacca ggccgtgaca gccaccggcc ggctgagcag cacagatcct
     5881 aacctgcaaa acatcccagt gcggaacgaa gagggcagaa gaatcagaca ggctttcatt
     5941 gcccctgagg actacgtcat cgtgtctgct gactacagcc agatagaact gagaatcatg
     6001 gcccatctgt ctcgggacaa gggcctgctg accgcttttg cagaaggcaa ggacatccac
     6061 agagccaccg ctgccgaagt gttcggcctg cctctggaaa cagtgaccag cgagcaaaga
     6121 aggtccgcca aggccattaa cttcggcctg atctacggca tgagcgcctt tggcctggcc
     6181 agacagctca acatccctag aaaggaggcc cagaaataca tggacctgta cttcgagcgg
     6241 taccccggcg tgctggagta catggaacgg accagagccc aggctaaaga gcagggatac
     6301 gtggaaaccc tggatggccg gagactgtat ctgcctgaca tcaagagcag caacggcgcc
     6361 agaagagccg ccgctgagag agctgctatc aacgccccta tgcagggcac cgccgccgac
     6421 atcatcaagc gggccatgat cgccgtggac gcctggctgc aggccgagca gcctagagtg
     6481 cggatgatca tgcaggtgca cgacgagctg gtgtttgagg tgcacaagga cgacgtggat
     6541 gctgtggcca agcagattca ccagctgatg gaaaattgca ccagactgga cgtgccactg
     6601 ctggtcgagg ttggaagcgg agagaattgg gaccaggccc actctggcgg ctcaaaaaga
     6661 accgccgacg gcagcgaatt cgagtctccc aagaagaaga ggaaagtctg accggtcatc
     6721 atcaccatca ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     6781 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     6841 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     6901 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     6961 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga
     7021 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     7081 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     7141 agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     7201 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     7261 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     7321 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     7381 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     7441 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     7501 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     7561 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     7621 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     7681 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     7741 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     7801 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     7861 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     7921 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     7981 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     8041 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     8101 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     8161 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     8221 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     8281 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     8341 gtgctgcaat gataccgcgc gaaccacgct caccggctcc agatttatca gcaataaacc
     8401 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     8461 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     8521 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     8581 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     8641 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     8701 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     8761 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     8821 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     8881 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     8941 gttcgatgta acccactcgt gcaccca
//
