LOCUS       sequence-220495-        3954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV-T7 promoter expression plasmid for human codon optimized AcrVA3 
            anti-CRISPR protein with C-terminal NLS .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3954)
  AUTHORS   Marino ND, Zhang JY, Borges AL, Sousa AA, Leon LM, Rauch BJ, Walton 
            RT, Berry JD, Joung JK, Kleinstiver BP, Bondy-Denomy J
  TITLE     Discovery of widespread Type I and Type V CRISPR-Cas inhibitors.
  JOURNAL   Science. 2018 Sep 6. pii: science.aau5174. doi: 
            10.1126/science.aau5174.
  PUBMED    30190308
REFERENCE   2  (bases 1 to 3954)
  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: "Science. 
            2018 Sep 6. pii: science.aau5174. doi: 10.1126/science.aau5174."
COMMENT     Sequence Label: pCMV-T7-hAcrVA3-NLS(sv40) (BPK5077)
FEATURES             Location/Qualifiers
     source          1..3954
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             532..552
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             561..578
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(601..618)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    607..831
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(902..918)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(902..918)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(915..937)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    926..942
                     /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(950..956,957..974,975..980))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    995..1016
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1133..1150)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1304..1892)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1384..1403)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2063..2854,2855..2923))
                     /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     2686..2705
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2924..3028)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3107..3126)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3298..3677
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3678..3881
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3832..3852
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3923..3942
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3923..3941
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      join(3949..3954,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atgggcgttg gtaaatcaaa aattgactgg caatctatag actggactaa aaccaatgcg
       61 caaattgccc aagaatgtgg gcgagcatat aacaccgtgt gcaaaatgcg gggtaaactt
      121 ggcaagagcc atcaaggcgc caaatctccg cgcaaggata aaggcatcag tcgccctcaa
      181 ccccatttga accggcttga gtaccaggca cttgccacag cgaaagcaaa ggccagccca
      241 aaagcgggac gattcgagac aaacacaaaa gcaaaaacat ggactctgaa atcacctgat
      301 aataaaacat atacgtttac gaatctgatg cactttgttc gcacaaaccc acatctgttt
      361 gaccccgatg atgtagtgtg gcggacaaaa agcaatggtg tggaatggtg cagggcctca
      421 agcgggctgg cgctgcttgc caaacgcaag aaagcgccac tttcttggaa aggttggcgc
      481 cttatttctc tcacaaaaga taacaaagga tccggcggtg gaggcagtgg gcccaagaag
      541 aagaggaaag tctgaccggt catcatcacc atcaccattg agtttaaacc cgctgatcag
      601 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
      661 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
      721 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
      781 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
      841 cggaaagaac cagctggggc tcgataccgt cgacctctag ctagagcttg gcgtaatcat
      901 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
      961 ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc acattaattg
     1021 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     1081 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     1141 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     1201 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     1261 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     1321 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     1381 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     1441 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     1501 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     1561 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     1621 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     1681 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     1741 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     1801 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     1861 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     1921 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     1981 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     2041 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     2101 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     2161 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     2221 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     2281 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     2341 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     2401 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     2461 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     2521 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     2581 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     2641 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     2701 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     2761 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     2821 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     2881 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     2941 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     3001 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     3061 acggatcggg agatcgatct cccgatcccc tagggtcgac tctcagtaca atctgctctg
     3121 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     3181 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     3241 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
     3301 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     3361 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     3421 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     3481 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     3541 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     3601 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     3661 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     3721 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     3781 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     3841 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga
     3901 tccgctagag atccgcggcc gctaatacga ctcactatag ggagagccgc cacc
//
