LOCUS       sequence-267384-        3750 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            AcrIIA17_Sga with C-terminal NLS (SV40).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3750)
  AUTHORS   Mahendra C, Christie KA, Osuna BA, Pinilla-Redondo R, Kleinstiver 
            BP, Bondy-Denomy J
  TITLE     Broad-spectrum anti-CRISPR proteins facilitate horizontal gene 
            transfer.
  JOURNAL   Nat Microbiol. 2020 Apr;5(4):620-629. doi: 
            10.1038/s41564-020-0692-2. Epub 2020 Mar 26.
  PUBMED    32218510
REFERENCE   2  (bases 1 to 3750)
  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"; doi: 
            "10.1038/s41564-020-0692-2"; journalName: "Nat Microbiol"; date: 
            "2020-04"; volume: "5"; issue: "4"; pages: "620-629"
COMMENT     Sequence Label: pCMV-T7-AcrIIA17_Sga-NLS(SV40) (KAC92)
FEATURES             Location/Qualifiers
     source          1..3750
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     17..36
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        17..35
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      43..52
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             376..396
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             405..422
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(445..462)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    451..675
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(746..762)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(746..762)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(759..781)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    770..786
                     /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(794..800,801..818,819..824))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    839..860
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(977..994)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1148..1736)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1228..1247)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1907..2698,2699..2767))
                     /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     2530..2549
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2768..2872)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2951..2970)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3142..3521
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3522..3725
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3676..3696
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gggcaagatc
       61 agcgtggaca gcgagaagct gctgaacgag gccatcaacg acttcgacat cttcggcgag
      121 gacttcaacg tgtacgccat ctacagctac agagaggact acgacttcga gtacatcagc
      181 gactacgtgg acgccgacga gcccaccaga gacgagttcg agaccgagga ggactaccag
      241 gaggtgatga aggacttcaa ggagaacctg gacagcctga agttcaccaa gcacaagaag
      301 atgaccatcg ccgacctggt gcacgagctg tgggagcaga acagaatctt cggatccggc
      361 ggtggaggca gtgggcccaa gaagaagagg aaagtctgac cggtcatcat caccatcacc
      421 attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
      481 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
      541 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
      601 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
      661 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgata ccgtcgacct
      721 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
      781 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag ggtgcctaat
      841 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
      901 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
      961 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     1021 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     1081 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     1141 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     1201 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     1261 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     1321 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     1381 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     1441 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     1501 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     1561 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     1621 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     1681 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     1741 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     1801 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     1861 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     1921 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     1981 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     2041 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     2101 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     2161 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     2221 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     2281 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     2341 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     2401 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     2461 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     2521 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     2581 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     2641 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     2701 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     2761 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     2821 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     2881 cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat cccctagggt
     2941 cgactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     3001 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     3061 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     3121 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     3181 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     3241 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     3301 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     3361 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     3421 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     3481 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     3541 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     3601 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     3661 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     3721 gagctggttt agtgaaccgt cagatccgct
//
