LOCUS       sequence-267380-        3774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            AcrIIA17_Efa with C-terminal NLS (SV40).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3774)
  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 3774)
  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-AcrIIA17_Efa-NLS(SV40) (KAC91)
FEATURES             Location/Qualifiers
     source          1..3774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     246..265
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        246..264
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      272..281
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             629..649
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             658..675
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(698..715)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    704..928
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(999..1015)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(999..1015)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1012..1034)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1023..1039
                     /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(1047..1053,1054..1071,1072..1077))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1092..1113
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1230..1247)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1401..1989)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1481..1500)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2160..2951,2952..3020))
                     /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     2783..2802
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3021..3125)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3204..3223)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3395..3774
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gagatccgcg
      241 gccgctaata cgactcacta tagggagagc cgccaccatg gccatcctga acaacaaggg
      301 cgagaagatc agcatcgact gcgccgacct gatcagcgag gtggaggagg acatcctgat
      361 cttcggcggc accttcctgg tgtacgccat ctgcagctgg agagagatcg agcaggtgga
      421 gtacatcagc gactacgtgc acgccgacaa ccccgagagc tacaaggacg agctgaccac
      481 caaggagtac gccgagctga aggagatcta cgagaaggac ctggaggagc tgaagatcac
      541 caagaacaag cagatgaacc tgaacgagct gctgagcatc ctgaccatcc agaacagcat
      601 caccggatcc ggcggtggag gcagtgggcc caagaagaag aggaaagtct gaccggtcat
      661 catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
      721 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
      781 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
      841 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
      901 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg
      961 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     1021 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     1081 tagggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     1141 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1201 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     1261 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     1321 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     1381 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     1441 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     1501 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     1561 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     1621 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     1681 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     1741 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     1801 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     1861 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     1921 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1981 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     2041 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     2101 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     2161 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     2221 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     2281 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     2341 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     2401 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     2461 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     2521 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     2581 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     2641 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     2701 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     2761 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     2821 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     2881 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     2941 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     3001 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     3061 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     3121 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcgatctccc
     3181 gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     3241 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
     3301 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
     3361 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
     3421 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     3481 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     3541 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     3601 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     3661 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     3721 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catg
//
