LOCUS       sequence-267382-        3822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            AcrIIA19_Ssim with C-terminal NLS (SV40).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3822)
  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 3822)
  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-AcrIIA19_Ssim-NLS(SV40) (KAC97)
FEATURES             Location/Qualifiers
     source          1..3822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     246..265
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        246..264
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      272..281
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             677..697
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             706..723
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(746..763)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    752..976
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1047..1063)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1047..1063)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1060..1082)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1071..1087
                     /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(1095..1101,1102..1119,1120..1125))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1140..1161
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1278..1295)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1449..2037)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1529..1548)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2208..2999,3000..3068))
                     /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     2831..2850
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3069..3173)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3252..3271)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3443..3822
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gagatccgcg
      241 gccgctaata cgactcacta tagggagagc cgccaccatg ggcaagctga tcgtggaggt
      301 ggaggagacc aactacaaga acctggtgaa ctacaccaag ctgaccaacg agagccacaa
      361 catcctggtg aacagactga tcagcgagta catcaccaag ccctacgagc tgagactgga
      421 cctgagcgag agatacagca acagagacct gatcgagttc aagttcatgc tgatcgagta
      481 ctgcaaggag gccctgcagg acatcaagga gctggccaac agcgacgagg cctacgagac
      541 cgacgaggcc ttcgaggccg tgttcagaca gctgttcgag gaggtgatca gcaaccccga
      601 caccgtgctg aaggccttcc acagctacac cagcttcctg gaggagaaca agggatccgg
      661 cggtggaggc agtgggccca agaagaagag gaaagtctga ccggtcatca tcaccatcac
      721 cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
      781 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
      841 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
      901 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
      961 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctcgat accgtcgacc
     1021 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     1081 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagccta gggtgcctaa
     1141 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     1201 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     1261 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     1321 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     1381 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     1441 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     1501 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     1561 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     1621 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     1681 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     1741 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     1801 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     1861 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     1921 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     1981 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     2041 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     2101 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     2161 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     2221 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     2281 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     2341 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     2401 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     2461 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     2521 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     2581 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     2641 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     2701 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     2761 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     2821 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     2881 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     2941 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     3001 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     3061 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     3121 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     3181 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc gatctcccga tcccctaggg
     3241 tcgactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     3301 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
     3361 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
     3421 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     3481 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     3541 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     3601 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     3661 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     3721 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     3781 acttggcagt acatctacgt attagtcatc gctattacca tg
//
