LOCUS       sequence-195015-        5230 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 5230)
  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 5230)
  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: AcrIIA4
FEATURES             Location/Qualifiers
     source          1..5230
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(185..190,191..206,207..213)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    221..237
                     /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             260..913
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     871..893
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             920..943
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1282..1304)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1449..1467)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1535..1639
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1640..1708,1709..2500)
                     /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(1858..1877)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2671..3259
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3160..3179
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3413..3430
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3503..3580
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3581..4663
                     /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(3600..3619)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4712..4717,4718..4735,4736..4742)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4750..4766
                     /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     4774..4790
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4774..4790
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4786..5230,1..83)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4806..4823)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4806..4822)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4815..4837)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttagcttatc gactgcacgg tgcaccaatg cttctggcgt caggcagcca tcggaagctg
       61 tggtatggct gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc
      121 gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga
      181 gctgttgaca attaatcatc ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt
      241 cacacaggaa acagtattca tgtcccctat actaggttat tggaaaatta agggccttgt
      301 gcaacccact cgacttcttt tggaatatct tgaagaaaaa tatgaagagc atttgtatga
      361 gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa
      421 tcttccttat tatattgatg gtgatgttaa attaacacag tctatggcca tcatacgtta
      481 tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat
      541 gcttgaagga gcggttttgg atattagata cggtgtttcg agaattgcat atagtaaaga
      601 ctttgaaact ctcaaagttg attttcttag caagctacct gaaatgctga aaatgttcga
      661 agatcgttta tgtcataaaa catatttaaa tggtgatcat gtaacccatc ctgacttcat
      721 gttgtatgac gctcttgatg ttgttttata catggaccca atgtgcctgg atgcgttccc
      781 aaaattagtt tgttttaaaa aacgtattga agctatccca caaattgata agtacttgaa
      841 atccagcaag tatatagcat ggcctttgca gggctggcaa gccacgtttg gtggtggcga
      901 ccatcctcca aaatcggatc tggaagttct gttccagggg cccctgggat ccatgaatat
      961 taatgactta attagagaaa tcaaaaacaa agattacaca gtgaaattga gtggtacgga
     1021 tagcaatagt atcacacagc taattattcg cgttaataat gatggcaacg agtatgtaat
     1081 ttctgaaagt gaaaatgaat caatcgttga aaaattcatc tctgcattca aaaacggttg
     1141 gaatcaagaa tacgaggatg aagaagaatt ttataatgac atgcaaacaa tcaccttaaa
     1201 aagtgagttg aactaactcg agcggccgca tcgtgactga ctgacgatct gcctcgcgcg
     1261 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     1321 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     1381 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata attcttgaag
     1441 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     1501 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1561 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     1621 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     1681 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     1741 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     1801 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     1861 atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca
     1921 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     1981 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     2041 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     2101 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     2161 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     2221 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     2281 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     2341 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     2401 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     2461 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     2521 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     2581 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     2641 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     2701 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     2761 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     2821 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     2881 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     2941 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3001 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3061 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3121 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3181 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3241 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3301 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3361 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3421 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3481 tatttcacac cgcataaatt ccgacaccat cgaatggcgc aaaacctttc gcggtatggc
     3541 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     3601 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     3661 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     3721 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     3781 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     3841 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     3901 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     3961 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     4021 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     4081 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     4141 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     4201 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     4261 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     4321 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     4381 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     4441 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     4501 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     4561 aaccacgcta gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     4621 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     4681 taagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     4741 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     4801 gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4861 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     4921 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg
     4981 gcaccagaag cggtgccgga aagctggctg gagtgcgatc ttcctgaggc cgatactgtc
     5041 gtcgtcccct caaactggca gatgcacggt tacgatgcgc ccatctacac caacgtgacc
     5101 tatcccatta cggtcaatcc gccgtttgtt cccacggaga atccgacggg ttgttactcg
     5161 ctcacattta atgttgatga aagctggcta caggaaggcc agacgcgaat tatttttgat
     5221 ggcgttggaa
//
