LOCUS       sequence-267378-        3996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            AcrIIA18_Sma with C-terminal NLS (SV40).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3996)
  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 3996)
  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-AcrIIA18_Sma-NLS(SV40) (KAC95)
FEATURES             Location/Qualifiers
     source          1..3996
                     /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             851..871
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             880..897
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(920..937)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    926..1150
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1221..1237)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1221..1237)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1234..1256)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1245..1261
                     /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(1269..1275,1276..1293,1294..1299))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1314..1335
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1452..1469)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1623..2211)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1703..1722)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2382..3173,3174..3242))
                     /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     3005..3024
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3243..3347)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3426..3445)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3617..3996
                     /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 ggcaagatcg acaccaccgt
      301 gaccgaggtg aaggagaacg gcaagaccta cctgagactg ctgaagggca acgagcagct
      361 gaaggccgtg agcgacaagg ccgtggccgg cgtgaacctg ttccccggcg ccaagatcgg
      421 cagcttcctg gtgagacagg acaacatcgt ggtgttcccc gacaacaagg gcgagttcga
      481 cctggacttc ttcaacctgc tgaacgacaa cttcgagacc ctggtggagt acgccaagat
      541 ggccgactgc ctggacatcg ccttcgacat caacgagaag agctacttca acatgatcat
      601 gtggctgatg aagaacatcg acgagaactg gagccagagc ccctacggcg agagcttcta
      661 cagcagcaag gacatcgact ggggctacaa gcccgagggc agcctgagag tgagcgacca
      721 ctggaacttc ggccaggacg gcgagcactg ccccaccgcc gagcccgtgg acggctgggc
      781 cgtgtgcaag ttcgagaacg gcaagtacca cctgatcaag aagttcggat ccggcggtgg
      841 aggcagtggg cccaagaaga agaggaaagt ctgaccggtc atcatcacca tcaccattga
      901 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
      961 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     1021 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     1081 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     1141 ggctctatgg cttctgaggc ggaaagaacc agctggggct cgataccgtc gacctctagc
     1201 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     1261 attccacaca acatacgagc cggaagcata aagtgtaaag cctagggtgc ctaatgagtg
     1321 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1381 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1441 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1501 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1561 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1621 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1681 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1741 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1801 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1861 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1921 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     1981 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2041 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2101 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2161 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2221 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2281 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2341 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2401 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2461 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2521 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2581 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2641 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2701 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2761 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     2821 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     2881 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     2941 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3001 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3061 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3121 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3181 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3241 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3301 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3361 aaagtgccac ctgacgtcga cggatcggga gatcgatctc ccgatcccct agggtcgact
     3421 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
     3481 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
     3541 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
     3601 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
     3661 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     3721 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     3781 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     3841 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     3901 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     3961 cagtacatct acgtattagt catcgctatt accatg
//
