LOCUS       sequence-190379-        5338 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Anti-CRISPRprotein  which inhibit SpyCas9 and LmoCas9 activity..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5338)
  AUTHORS   Dong D, Guo M, Wang S, Zhu Y, Wang S, Xiong Z, Yang J, Xu Z, Huang Z
  TITLE     Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR 
            protein.
  JOURNAL   Nature. 2017 Jun 15;546(7658):436-439. doi: 10.1038/nature22377. 
            Epub 2017 Apr 27.
  PUBMED    28448066
REFERENCE   2  (bases 1 to 5338)
  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/nature22377"; journalName: "Nature"; date: "2017-06-15- 
            15"; volume: "546"; issue: "7658"; pages: "436-439"
COMMENT     Sequence Label: AcrIIA2
FEATURES             Location/Qualifiers
     source          1..5338
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1388..1410)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1555..1573)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1641..1745
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1746..1814,1815..2606)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1964..1983)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2777..3365
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3266..3285
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3519..3536
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3609..3686
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3687..4769
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3706..3725)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    4782..4803
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4818..4823,4824..4841,4842..4848)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4856..4872
                     /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)."
     primer_bind     4880..4896
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4880..4896
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4892..5338,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4912..4929)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4912..4928)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4921..4943)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc atgacattaa
      961 caagagcaca aaaaaagtat gcagaagcga tgcacgagtt tataaatatg gtggatgact
     1021 ttgaagagtc tacaccggat tttgcaaagg aagttctaca tgattctgac tatgtagtta
     1081 ttacaaaaaa tgaaaaatat gcagtagctc tttgctctct tagcactgat gaatgtgaat
     1141 atgatactaa cttatactta gatgaaaaat tggttgatta ctcaacagtt gacgtaaacg
     1201 gtgtgacata ctacatcaac attgttgaaa ctaacgatat cgatgattta gaaatcgcta
     1261 cggatgaaga tgagatgaaa agtggcaacc aagaaattat tttaaaaagt gagttgaagt
     1321 aactcgagcg gccgcatcgt gactgactga cgatctgcct cgcgcgtttc ggtgatgacg
     1381 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     1441 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     1501 ccatgaccca gtcacgtagc gatagcggag tgtataattc ttgaagacga aagggcctcg
     1561 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     1621 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1681 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     1741 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     1801 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     1861 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     1921 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     1981 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2041 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2101 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     2161 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     2221 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     2281 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     2341 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     2401 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     2461 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     2521 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     2581 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     2641 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     2701 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2761 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2821 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2881 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     2941 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3001 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3061 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3121 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3181 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3241 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3301 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3361 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3421 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3481 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3541 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3601 taaattccga caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     3661 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
     3721 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     3781 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     3841 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     3901 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     3961 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     4021 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     4081 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     4141 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
     4201 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     4261 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
     4321 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
     4381 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     4441 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg
     4501 gatacgacga taccgaagac agctcatgtt atatcccgcc gtcaaccacc atcaaacagg
     4561 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     4621 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     4681 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     4741 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     4801 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     4861 agcggataac aatttcacac aggaaacagc tatgaccatg attacggatt cactggccgt
     4921 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     4981 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     5041 acagttgcgc agcctgaatg gcgaatggcg ctttgcctgg tttccggcac cagaagcggt
     5101 gccggaaagc tggctggagt gcgatcttcc tgaggccgat actgtcgtcg tcccctcaaa
     5161 ctggcagatg cacggttacg atgcgcccat ctacaccaac gtaacctatc ccattacggt
     5221 caatccgccg tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt
     5281 tgatgaaagc tggctacagg aaggccagac gcgaattatt tttgatggcg ttggaatt
//
