LOCUS       sequence-220494-        4416 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV-T7 promoter expression plasmid for human codon optimized AcrVA2 
            anti-CRISPR protein with C-terminal NLS .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4416)
  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 4416)
  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-NLS(sv40) (AAS2283)
FEATURES             Location/Qualifiers
     source          1..4416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             994..1014
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1023..1040
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1063..1080)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1069..1293
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1364..1380)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1364..1380)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1377..1399)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1388..1404
                     /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(1412..1418,1419..1436,1437..1442))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1457..1478
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1595..1612)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1766..2354)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1846..1865)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2525..3316,3317..3385))
                     /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     3148..3167
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3386..3490)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3569..3588)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3760..4139
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4140..4343
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4294..4314
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4385..4404
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4385..4403
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      join(4411..4416,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atgggccatc acaccatcgc ccgcatgaat gctttcaaca aggcttttgc aaacgctaaa
       61 gactgctaca aaaaaatgca agcctggcac ctgttgaaca aaccgaaaca cgcattcttt
      121 cctatgcaga acacgccagc cctggataac ggcctcgctg cgctttatga actccggggg
      181 ggaaaggaag atgcacatat tttgtctatt ctcagcagat tgtacttgta cggagcgtgg
      241 cggaatacat tgggcatata ccagttggac gaggagatca ttaaggactg caaggagctt
      301 cccgacgata caccgacgag catattcctc aacttgcctg actggtgtgt atatgtagat
      361 atatccagtg ctcagatcgc cactttcgac gacggcgtag cgaagcacat caaaggtttc
      421 tgggccatat acgatattgt tgaaatgaat ggaatcaacc atgacgtatt ggacttcgtt
      481 gtagatactg acaccgacga taatgtgtac gttccgcagc ctttcatttt gtcatctggt
      541 caatccgtgg cagaagtgct tgactatgga gcaagtttgt tcgacgacga tactagcaat
      601 acgcttatta agggcctcct gccgtacttg ctctggcttt gcgtagcaga acccgacatc
      661 acttacaaag gtttgccagt ctcaagagag gaacttactc gaccgaaaca ttcaatcaat
      721 aaaaaaacgg gcgcgtttgt gacacctagt gagccattca tctaccagat aggagaacgg
      781 ctgggaagtg aggttaggag gtatcagagc attatagatg gggagcaaaa gcgcaatcga
      841 ccacatacaa agcgccctca tattcgccgc ggtcactggc atggatattg gcaagggact
      901 ggacaggcga aggagtttcg agttcgatgg caacccgctg tgttcgtgaa tagcggaaga
      961 gtatcatccg gatccggcgg tggaggcagt gggcccaaga agaagaggaa agtctgaccg
     1021 gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact gtgccttcta
     1081 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     1141 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     1201 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     1261 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     1321 gctcgatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     1381 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     1441 aagcctaggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     1501 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     1561 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     1621 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     1681 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     1741 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1801 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1861 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1921 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1981 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2041 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2101 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2161 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2221 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2281 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2341 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2401 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2461 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2521 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2581 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2641 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     2701 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     2761 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     2821 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2881 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2941 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3001 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3061 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3121 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3181 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3241 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3301 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3361 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3421 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3481 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatcgat
     3541 ctcccgatcc cctagggtcg actctcagta caatctgctc tgatgccgca tagttaagcc
     3601 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     3661 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
     3721 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
     3781 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     3841 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     3901 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     3961 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     4021 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     4081 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     4141 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     4201 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     4261 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     4321 gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag agatccgcgg
     4381 ccgctaatac gactcactat agggagagcc gccacc
//
