LOCUS       sequence-267367-        3993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            AcrIIA18_Sga with C-terminal NLS (SV40).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3993)
  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 3993)
  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_Sga-NLS(SV40) (KAC214)
FEATURES             Location/Qualifiers
     source          1..3993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      652..661
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1228..1248
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1257..1274
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1297..1314)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1303..1527
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1598..1614)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1598..1614)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1611..1633)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1622..1638
                     /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(1646..1652,1653..1670,1671..1676))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1691..1712
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1829..1846)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2000..2588)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2080..2099)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2759..3550,3551..3619))
                     /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     3382..3401
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3620..3724)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3803..3822)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gagatccgcg gccgctaata cgactcacta tagggagagc cgccaccatg
      661 ggcaagatcg acaccaccgt gaccgaggtg aaggagaacg gcaagaccta cctgagactg
      721 gtggagggca ccgagcagct gaaggccatc agcgacaagg ccatggccgg cgtgaacctg
      781 ttccccggcg ccaagatcga cagcttcctg gtgaagcagg acagcatcgt ggtgttcccc
      841 gacaacaagg gcgagttcga cctggacttc ttcaagcagc tggacgagaa cttcgacacc
      901 atcgccaagt acgccagagt ggccacctgc ttcgaggagg tggccttcga cgagaagagc
      961 tacttcaaca tgatcatgtg gctgatggac aacatggacg agaactggag ccagagcccc
     1021 tacggcgaga gcttctacag cagcaagaac atcgactggg gctacaagcc cgagggcagc
     1081 ctgagagtga gcgaccactg gaacttcggc gagaacggcg agcactgccc caccgccgag
     1141 cccgtggacg gctgggccgt gtgcaagttc gagaacggca agtaccacct gatcaagaag
     1201 ttcggatccg gcggtggagg cagtgggccc aagaagaaga ggaaagtctg accggtcatc
     1261 atcaccatca ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     1321 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     1381 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     1441 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     1501 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga
     1561 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     1621 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     1681 agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1741 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1801 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     1861 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     1921 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     1981 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2041 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2101 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2161 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2221 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2281 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2341 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2401 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2461 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2521 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2581 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     2641 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2701 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     2761 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2821 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2881 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     2941 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3001 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3061 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3121 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3181 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3241 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3301 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3361 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3421 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3481 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3541 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     3601 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     3661 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     3721 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg
     3781 atcccctagg gtcgactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     3841 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     3901 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     3961 tgcttcgcga tgtacgggcc agatatacgc gtt
//
