LOCUS       sequence-195009-        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(187..192,193..208,209..215)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    223..239
                     /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             262..915
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     873..895
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             922..945
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1392..1414)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1559..1577)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1645..1749
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1750..1818,1819..2610)
                     /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(1968..1987)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2781..3369
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3270..3289
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3523..3540
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3613..3690
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3691..4773
                     /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
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3710..3729)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4822..4827,4828..4845,4846..4852)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4860..4876
                     /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     4884..4900
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4884..4900
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4896..5338,1..85)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4916..4933)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4916..4932)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4925..4947)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 aattagctta tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc
       61 tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca aggcgcactc
      121 ccgttctgga taatgttttt tgcgccgaca tcataacggt tctggcaaat attctgaaat
      181 gagctgttga caattaatca tcggctcgta taatgtgtgg aattgtgagc ggataacaat
      241 ttcacacagg aaacagtatt catgtcccct atactaggtt attggaaaat taagggcctt
      301 gtgcaaccca ctcgacttct tttggaatat cttgaagaaa aatatgaaga gcatttgtat
      361 gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc
      421 aatcttcctt attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt
      481 tatatagctg acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca
      541 atgcttgaag gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa
      601 gactttgaaa ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc
      661 gaagatcgtt tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc
      721 atgttgtatg acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc
      781 ccaaaattag tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg
      841 aaatccagca agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc
      901 gaccatcctc caaaatcgga tctggaagtt ctgttccagg ggcccctggg atccatgaca
      961 ttaacaagag cacaaaaaaa gtatgcagaa gcgatgcacg agtttataaa tatggtggat
     1021 gactttgaag agtctacacc ggattttgca aaggaagttc tacatgattc tgactatgta
     1081 gttattacaa aaaatgaaaa atatgcagta gctctttgct ctcttagcac tgatgaatgt
     1141 gaatatgata ctaacttata cttagatgaa aaattggttg attactcaac agttgacgta
     1201 aacggtgtga catactacat caacattgtt gaaactaacg atatcgatga tttagaaatc
     1261 gctacggatg aagatgagat gaaaagtggc aaccaagaaa ttattttaaa aagtgagttg
     1321 aagtaactcg agcggccgca tcgtgactga ctgacgatct gcctcgcgcg tttcggtgat
     1381 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     1441 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     1501 gcagccatga cccagtcacg tagcgatagc ggagtgtata attcttgaag acgaaagggc
     1561 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     1621 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1681 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1741 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1801 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1861 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1921 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1981 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2041 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2101 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2161 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2221 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2281 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2341 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2401 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2461 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2521 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2581 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2641 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2701 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2761 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2821 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2881 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2941 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3001 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3061 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3121 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3181 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3241 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3301 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3361 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3421 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3481 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3541 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3601 cgcataaatt ccgacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
     3661 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     3721 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     3781 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     3841 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     3901 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     3961 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     4021 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     4081 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     4141 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     4201 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     4261 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     4321 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     4381 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     4441 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     4501 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     4561 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     4621 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccacgcta
     4681 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     4741 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     4801 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     4861 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg gattcactgg
     4921 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     4981 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     5041 cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg gcaccagaag
     5101 cggtgccgga aagctggctg gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct
     5161 caaactggca gatgcacggt tacgatgcgc ccatctacac caacgtgacc tatcccatta
     5221 cggtcaatcc gccgtttgtt cccacggaga atccgacggg ttgttactcg ctcacattta
     5281 atgttgatga aagctggcta caggaaggcc agacgcgaat tatttttgat ggcgttgg
//
