LOCUS       sequence-220492-        4407 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV-T7 promoter expression plasmid for human codon optimized 
            AcrVA2.1 anti-CRISPR protein with C-terminal NLS .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4407)
  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 4407)
  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-hAcrVA2.1-NLS(sv40) (BPK5059)
FEATURES             Location/Qualifiers
     source          1..4407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             985..1005
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1014..1031
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1054..1071)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1060..1284
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1355..1371)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1355..1371)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1368..1390)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1379..1395
                     /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(1403..1409,1410..1427,1428..1433))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1448..1469
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1586..1603)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1757..2345)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1837..1856)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2516..3307,3308..3376))
                     /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     3139..3158
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3377..3481)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3560..3579)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3751..4130
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4131..4334
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4285..4305
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4376..4395
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4376..4394
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      join(4402..4407,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atgggccacc acacgatagc tagaatgaac gcctttaaca aggctttcgg caatgccaag
       61 gactgttaca agaaaatgca ggcctggcac ctcaataaca aaccaaaaca tatctttagc
      121 ccgctccaga ataccctctc actgaatgaa ggcttggcag cgctttatga gcttcatggc
      181 gggaaggagg atgaacacat tctgtctatc ctctgttgcc tgtatctgta tgggacatgg
      241 agaaacacac tgggcatata ccaactggat gaagaaataa ttaaggattg caaagagttg
      301 cccgatgaca cacctacaag cattttcctt aacttgcctg actggtgtgt ttacgtcgat
      361 atctcaagcg cgaaaattgc caccatagat ggcggcgtcg caaagcacat aaagggtttt
      421 tgggcgatct acgacaacat tgagatgcac ggcgtgaacc acgatgtcct caattttata
      481 atcgacactg atacggacaa caatatctat gtaccacagt ctcttatcct cagttctgag
      541 atgtcagtag cggagagtct tgactacggt ctgaccctct tcgggtatga cgaatctaac
      601 gagttggtca aagggatgct tccatacctc ctttggctgt gcgttgctga gccggacata
      661 acacacaaag gcttgccagt cagcagagag gagcttacaa agcctaaaca cggaataaat
      721 aagaagacgg gggccttcgt gacgcctagc gaacctttca tatatcagat aggagaacgg
      781 ctcggtgggg aggtgagaag ataccagagc ctgatagacg atgaaaagaa ccaaaataga
      841 catcacacta aaaggccgca cataaggagg gggcactggc acggctactg gcaggggacg
      901 ggacaggcaa aggagtttaa ggttcgatgg caaccggctg tttttgtaaa ctcaggtgtc
      961 ggatccggcg gtggaggcag tgggcccaag aagaagagga aagtctgacc ggtcatcatc
     1021 accatcacca ttgagtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     1081 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     1141 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     1201 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     1261 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgatac
     1321 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     1381 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctagg
     1441 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1501 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1561 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1621 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1681 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1741 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1801 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1861 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1921 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1981 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2041 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2101 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2161 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2221 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2281 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2341 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2401 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2461 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2521 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2581 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2641 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     2701 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2761 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     2821 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     2881 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     2941 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3001 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3061 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3121 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3181 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3241 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3301 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3361 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     3421 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3481 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatcga tctcccgatc
     3541 ccctagggtc gactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     3601 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     3661 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     3721 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
     3781 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     3841 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     3901 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     3961 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     4021 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     4081 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     4141 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     4201 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     4261 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     4321 atataagcag agctggttta gtgaaccgtc agatccgcta gagatccgcg gccgctaata
     4381 cgactcacta tagggagagc cgccacc
//
