LOCUS       sequence-275302-        3897 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            AcrIIA1(T114A/F115A) with C-terminal NLS (SV40).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3897)
  AUTHORS   Osuna BA, Karambelkar S, Mahendra C, Christie KA, Garcia B, Davidson
            AR, Kleinstiver BP, Kilcher S, Bondy-Denomy J
  TITLE     Listeria Phages Induce Cas9 Degradation to Protect Lysogenic 
            Genomes.
  JOURNAL   Cell Host Microbe. 2020 Apr 18. pii: S1931-3128(20)30190-6. doi: 
            10.1016/j.chom.2020.04.001.
  PUBMED    32325050
REFERENCE   2  (bases 1 to 3897)
  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: "Cell Host 
            Microbe. 2020 Apr 18. pii: S1931-3128(20)30190-6. doi: 
            10.1016/j.chom.2020.04.001."
COMMENT     Sequence Label: pCMV-T7-AcrIIA1(T114A/F115A)-NLS(SV40) (KAC688)
FEATURES             Location/Qualifiers
     source          1..3897
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      403..412
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             883..903
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             912..929
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(952..969)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    958..1182
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1253..1269)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1253..1269)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1266..1288)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1277..1293
                     /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(1301..1307,1308..1325,1326..1331))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1346..1367
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1484..1501)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1655..2243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1735..1754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2414..3205,3206..3274))
                     /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     3037..3056
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3275..3379)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3458..3477)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(3649..3897,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gggcaccatc
      421 aagctgctgg acgagttcct gaagaagcac gacctgacca gataccagct gagcaagctg
      481 accggcatca gccagaacac cctgaaggac cagaacgaga agcccctgaa caagtacacc
      541 gtgagcatcc tgagaagcct gagcctgatc agcggcctga gcgtgagcga cgtgctgttc
      601 gagctggagg acatcgagaa gaacagcgac gacctggccg gcttcaagca cctgctggac
      661 aagtacaagc tgagcttccc cgcccaggag ttcgagctgt actgcctgat caaggagttc
      721 gagagcgcca acatcgaggt gctgcccttc gccgccaaca gattcgagaa cgaggagcac
      781 gtgaacatca agaaggacgt gtgcaaggcc ctggagaacg ccatcaccgt gctgaaggag
      841 aagaagaacg agctgctggg atccggcggt ggaggcagtg ggcccaagaa gaagaggaaa
      901 gtctgaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg
      961 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     1021 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     1081 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     1141 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     1201 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     1261 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     1321 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     1381 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     1441 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     1501 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     1561 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     1621 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     1681 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     1741 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     1801 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct
     1861 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     1921 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     1981 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2041 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     2101 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2161 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     2221 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     2281 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     2341 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     2401 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     2461 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     2521 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     2581 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     2641 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     2701 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     2761 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     2821 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     2881 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     2941 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     3001 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     3061 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3121 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     3181 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     3241 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     3301 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     3361 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     3421 gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat
     3481 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     3541 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     3601 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     3661 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     3721 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     3781 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     3841 gtcaatgggt ggactattta cggtaaactg cccacttggc agtacatcaa gtgtatc
//
