LOCUS       sequence-292636-        4571 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses type II-A anti-CRISPR protein AcrIIA5 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4571)
  AUTHORS   Garcia B, Lee J, Edraki A, Hidalgo-Reyes Y, Erwood S, Mir A, Trost 
            CN, Seroussi U, Stanley SY, Cohn RD, Claycomb JM, Sontheimer EJ, 
            Maxwell KL, Davidson AR
  TITLE     Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for 
            Genome Editing.
  JOURNAL   Cell Rep. 2019 Nov 12;29(7):1739-1746.e5. doi: 
            10.1016/j.celrep.2019.10.017.
  PUBMED    31722192
REFERENCE   2  (bases 1 to 4571)
  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: 
            "10.1016/j.celrep.2019.10"; journalName: "Cell Rep"; date: 
            "2019-11-12- 12"; volume: "29"; issue: "7"; pages: "1739-1746.e5"
COMMENT     Sequence Label: pEJS1004-pCSDest2-AcrIIA5-NLS-FLAG
FEATURES             Location/Qualifiers
     source          1..4571
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        845..863
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     845..862
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      910..919
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1342..1365
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1372..1392
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1433..1567
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1457..1476)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1511..1530
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1680..1700)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1680..1698)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1719..1735)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1719..1735)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1732..1754)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1743..1759
                     /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(1767..1773,1774..1791,1792..1797))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1812..1833
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1950..1967)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2121..2709)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2201..2220)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2880..3671,3672..3740))
                     /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     3503..3522
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3741..3845)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3871..4326)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4008..4029)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4220..4239
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(4397..4571,1..810)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 acctggcact gtgccaactg gggaggggtc tacttggcac ggtgccaagt ttgaggaggg
       61 gtcttggccc tgtgccaagt ccgccatatt gaattggcat ggtgccaata atggcggcca
      121 tattggctat atgccaggat caatatatag gcaatatcca atatggccct atgccaatat
      181 ggctattggc caggttcaat actatgtatt ggccctatgc catatagtat tccatatatg
      241 ggttttccta ttgacgtaga tagcccctcc caatgggcgg tcccatatac catatatggg
      301 gcttcctaat accgcccata gccactcccc cattgacgtc aatggtctct atatatggtc
      361 tttcctattg acgtcatatg ggcggtccta ttgacgtata tggcgcctcc cccattgacg
      421 tcaattacgg taaatggccc gcctggctca atgcccattg acgtcaatag gaccacccac
      481 cattgacgtc aatgggatgg ctcattgccc attcatatcc gttctcacgc cccctattga
      541 cgtcaatgac ggtaaatggc ccacttggca gtacatcaat atctattaat agtaacttgg
      601 caagtacatt actattggaa gtacgccagg gtacattggc agtactccca ttgacgtcaa
      661 tggcggtaaa tggcccgcga tggctgccaa gtacatcccc attgacgtca atggggaggg
      721 gcaatgacgc aaatgggcgt tccattgacg taaatgggcg gtaggcgtgc ctaatgggag
      781 gtctatataa gcaatgctcg tttagggaac cgccattctg cctggggacg tcggagcaag
      841 cttgatttag gtgacactat agaatacaag ctacttgttc tttttgcagg atcccatcga
      901 ttcgaagccg ccaccatggc ttatggtaaa tctcggtata attcttatag gaaaaggagc
      961 ttcaatcgct ctaataaaca acgccgggag tacgctcagg aaatggacag actggagaag
     1021 gcgtttgaga atcttgatgg atggtacttg tcatcaatga aagactcagc ctacaaagac
     1081 tttgggaagt atgagatccg gctgtctaac cacagcgcag ataacaagta ccatgatctc
     1141 gaaaatggac gactcattgt caatatcaaa gcttcaaagc tgaacttcgt cgatattata
     1201 gagaacaaac tggacaaaat catagagaaa atcgacaaat tggatctcga caagtatcga
     1261 tttataaatg caacgaatct tgaacatgac atcaaatgtt attacaaggg tttcaaaact
     1321 aaaaaggagg tcattggtgg tgattataag gatgacgatg acaaaggcag cccgaagaag
     1381 aaaaggaagg tgtgactcga gcctctagaa ctatagtgag tcgtattacg tagatccaga
     1441 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     1501 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     1561 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     1621 ggttttttaa ttcgcggccg cggcgccaat gcattgggcc cggtacccag cttttgttcc
     1681 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     1741 aattgttatc cgctcacaat tccacacaac atacgagccg ggagcataaa gtgtaaagcc
     1801 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     1861 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1921 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     1981 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2041 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2101 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2161 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2221 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2281 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2341 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2401 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2461 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2521 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     2581 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     2641 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     2701 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     2761 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     2821 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     2881 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     2941 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3001 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3061 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3121 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3181 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3241 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3301 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     3361 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     3421 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3481 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3541 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     3601 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     3661 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     3721 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     3781 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     3841 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
     3901 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     3961 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     4021 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     4081 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     4141 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     4201 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     4261 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     4321 gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     4381 cttcgctatt acgccagtcg accgccaatt caatatggcg tatatggact catgccaatt
     4441 caatatggtg gatctggacc tgtgccaatt caatatggcg tatatggact cgtgccaatt
     4501 caatatggtg gatctggacc ccagccaatt caatatggcg gacttggcac catgccaatt
     4561 caatatggcg g
//
