LOCUS       sequence-370317-        7595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes catalytically inactive Cas12a (D917A, E1006A) from F. 
            novicida (type V-A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7595)
  AUTHORS   Wu WY, Jackson SA, Almendros C, Haagsma AC, Yilmaz S, Gort G, van 
            der Oost J, Brouns SJJ, Staals RHJ
  TITLE     Adaptation by Type V-A and V-B CRISPR-Cas Systems Demonstrates 
            Conserved Protospacer Selection Mechanisms Between Diverse 
            CRISPR-Cas Types.
  JOURNAL   CRISPR J. 2022 Aug;5(4):536-547. doi: 10.1089/crispr.2021.0150. Epub
            2022 Jul 12.
  PUBMED    35833800
REFERENCE   2  (bases 1 to 7595)
  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: 
            "10.1089/crispr.2021.0150"; journalName: "CRISPR J"; date: 
            "2022-08"; volume: "5"; issue: "4"; pages: "536-547"
COMMENT     Sequence Label: pdCas12a(RuvC)
FEATURES             Location/Qualifiers
     source          1..7595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(565..1647)
                     /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     1609..1628
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1648..1725)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1771..1790
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1771..1789
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1790..1814
                     /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     complement(5820..5838)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5834..5881
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(6102..6761)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6676..6695
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6762..6864)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(7256..7273)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(7390..7595,1..340))
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(7470..7489)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc
       61 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca
      121 ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta
      181 aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt
      241 caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt
      301 ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca
      361 gataaaatat ttctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag
      421 ccccatacga tataagttgt aattctcatg ttagtcatgc cccgcgccca ccggaaggag
      481 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
      541 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
      601 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
      661 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
      721 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
      781 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
      841 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
      901 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
      961 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     1021 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     1081 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     1141 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     1201 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     1261 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     1321 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     1381 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     1441 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     1501 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     1561 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     1621 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     1681 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     1741 ctcccttatg cgactcctgc attaggaaat taatacgact cactataggg gaattgtgag
     1801 cggataacaa ttcccctgta gaaataattt tgtttaactt taataaggag atataccatg
     1861 gatgtcaatt tatcaagaat ttgttaataa atatagttta agtaaaactc taagatttga
     1921 gttaatccca cagggtaaaa cacttgaaaa cataaaagca agaggtttga ttttagatga
     1981 tgagaaaaga gctaaagact acaaaaaggc taaacaaata attgataaat atcatcagtt
     2041 ttttatagag gagatattaa gttcggtttg tattagcgaa gatttattac aaaactattc
     2101 tgatgtttat tttaaactta aaaagagtga tgatgataat ctacaaaaag attttaaaag
     2161 tgcaaaagat acgataaaga aacaaatatc tgaatatata aaggactcag agaaatttaa
     2221 gaatttgttt aatcaaaacc ttatcgatgc taaaaaaggg caagagtcag atttaattct
     2281 atggctaaag caatctaagg ataatggtat agaactattt aaagccaata gtgatatcac
     2341 agatatagat gaggcgttag aaataatcaa atcttttaaa ggttggacaa cttattttaa
     2401 gggttttcat gaaaatagaa aaaatgttta tagtagcaat gatattccta catctattat
     2461 ttataggata gtagatgata atttgcctaa atttctagaa aataaagcta agtatgagag
     2521 tttaaaagac aaagctccag aagctataaa ctatgaacaa attaaaaaag atttggcaga
     2581 agagctaacc tttgatattg actacaaaac atctgaagtt aatcaaagag ttttttcact
     2641 tgatgaagtt tttgagatag caaactttaa taattatcta aatcaaagtg gtattactaa
     2701 atttaatact attattggtg gtaaatttgt aaatggtgaa aatacaaaga gaaaaggtat
     2761 aaatgaatat ataaatctat actcacagca aataaatgat aaaacactca aaaaatataa
     2821 aatgagtgtt ttatttaagc aaattttaag tgatacagaa tctaaatctt ttgtaattga
     2881 taagttagaa gatgatagtg atgtagttac aacgatgcaa agtttttatg agcaaatagc
     2941 agcttttaaa acagtagaag aaaaatctat taaagaaaca ctatctttat tatttgatga
     3001 tttaaaagct caaaaacttg atttgagtaa aatttatttt aaaaatgata aatctcttac
     3061 tgatctatca caacaagttt ttgatgatta tagtgttatt ggtacagcgg tactagaata
     3121 tataactcaa caaatagcac ctaaaaatct tgataaccct agtaagaaag agcaagaatt
     3181 aatagccaaa aaaactgaaa aagcaaaata cttatctcta gaaactataa agcttgcctt
     3241 agaagaattt aataagcata gagatataga taaacagtgt aggtttgaag aaatacttgc
     3301 aaactttgcg gctattccga tgatatttga tgaaatagct caaaacaaag acaatttggc
     3361 acagatatct atcaaatatc aaaatcaagg taaaaaagac ctacttcaag ctagtgcgga
     3421 agatgatgtt aaagctatca aggatctttt agatcaaact aataatctct tacataaact
     3481 aaaaatattt catattagtc agtcagaaga taaggcaaat attttagaca aggatgagca
     3541 tttttatcta gtatttgagg agtgctactt tgagctagcg aatatagtgc ctctttataa
     3601 caaaattaga aactatataa ctcaaaagcc atatagtgat gagaaattta agctcaattt
     3661 tgagaactcg actttggcta atggttggga taaaaataaa gagcctgaca atacggcaat
     3721 tttatttatc aaagatgata aatattatct gggtgtgatg aataagaaaa ataacaaaat
     3781 atttgatgat aaagctatca aagaaaataa aggcgagggt tataaaaaaa ttgtttataa
     3841 acttttacct ggcgcaaata aaatgttacc taaggttttc ttttctgcta aatctataaa
     3901 attttataat cctagtgaag atatacttag aataagaaat cattccacac atacaaaaaa
     3961 tggtagtcct caaaaaggat atgaaaaatt tgagtttaat attgaagatt gccgaaaatt
     4021 tatagatttt tataaacagt ctataagtaa gcatccggag tggaaagatt ttggatttag
     4081 attttctgat actcaaagat ataattctat agatgaattt tatagagaag ttgaaaatca
     4141 aggctacaaa ctaacttttg aaaatatatc agagagctat attgatagcg tagttaatca
     4201 gggtaaattg tacctattcc aaatctataa taaagatttt tcagcttata gcaaagggcg
     4261 accaaatcta catactttat attggaaagc gctgtttgat gagagaaatc ttcaagatgt
     4321 ggtttataag ctaaatggtg aggcagagct tttttatcgt aaacaatcaa tacctaaaaa
     4381 aatcactcac ccagctaaag aggcaatagc taataaaaac aaagataatc ctaaaaaaga
     4441 gagtgttttt gaatatgatt taatcaaaga taaacgcttt actgaagata agtttttctt
     4501 tcactgtcct attacaatca attttaaatc tagtggagct aataagttta atgatgaaat
     4561 caatttattg ctaaaagaaa aagcaaatga tgttcatata ttaagtatag ccagaggtga
     4621 aagacattta gcttactata ctttggtaga tggtaaaggc aatatcatca aacaagatac
     4681 tttcaacatc attggtaatg atagaatgaa aacaaactac catgataagc ttgctgcaat
     4741 agagaaagat agggattcag ctaggaaaga ctggaaaaag ataaataaca tcaaagagat
     4801 gaaagagggc tatctatctc aggtagttca tgaaatagct aagctagtta tagagtataa
     4861 tgctattgtg gtttttgccg atttaaattt tggatttaaa agagggcgtt tcaaggtaga
     4921 gaagcaggtc tatcaaaagt tagaaaaaat gctaattgag aaactaaact atctagtttt
     4981 caaagataat gagtttgata aaactggggg agtgcttaga gcttatcagc taacagcacc
     5041 ttttgagact tttaaaaaga tgggtaaaca aacaggtatt atctactatg taccagctgg
     5101 ttttacttca aaaatttgtc ctgtaactgg ttttgtaaat cagttatatc ctaagtatga
     5161 aagtgtcagc aaatctcaag agttctttag taagtttgac aagatttgtt ataaccttga
     5221 taagggctat tttgagttta gttttgatta taaaaacttt ggtgacaagg ctgccaaagg
     5281 caagtggact atagctagct ttgggagtag attgattaac tttagaaatt cagataaaaa
     5341 tcataattgg gatactcgag aagtttatcc aactaaagag ttggagaaat tgctaaaaga
     5401 ttattctatc gaatatgggc atggcgaatg tatcaaagca gctatttgcg gtgagagcga
     5461 caaaaagttt tttgctaagc taactagtgt cctaaatact atcttacaaa tgcgtaactc
     5521 aaaaacaggt actgagttag attatctaat ttcaccagta gcagatgtaa atggcaattt
     5581 ctttgattcg cgacaggcgc caaaaaatat gcctcaagat gctgatgcca atggtgctta
     5641 tcatattggg ctaaaaggtc tgatgctact aggtaggatc aaaaataatc aagagggcaa
     5701 aaaactcaat ttggttatca aaaatgaaga gtattttgag ttcgtgcaga ataggaataa
     5761 ctaacccaga gaattcgagc tcggcgcgcc tgcaggtcga caagcttgcg ctgctgccac
     5821 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     5881 gctgaaacct caggcatttg agaagcacac ggtcacactg cttccggtag tcaataaacc
     5941 ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac cgacgaccgg
     6001 gtcgaatttg ctttcgaatt tctgccattc atccgcttat tatcacttat tcaggcgtag
     6061 caaccaggcg tttaagggca ccaataactg ccttaaaaaa attacgcccc gccctgccac
     6121 tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg
     6181 gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg
     6241 cccatagtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg
     6301 gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg
     6361 aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc
     6421 cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa
     6481 acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata
     6541 cggaactccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac
     6601 ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg
     6661 ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg
     6721 gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct
     6781 gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag
     6841 ttggaacctc ttacgtgccg atcaacgtct cattttcgcc aaaagttggc ccagggcttc
     6901 ccggtatcaa cagggacacc aggatttatt tattctgcga agtgatcttc cgtcacaggt
     6961 atttattcgg cgcaaagtgc gtcgggtgat gctgccaact tactgattta gtgtatgatg
     7021 gtgtttttga ggtgctccag tggcttctgt ttctatcagc tgtccctcct gttcagctac
     7081 tgacggggtg gtgcgtaacg gcaaaagcac cgccggacat cagcgctagc ggagtgtata
     7141 ctggcttact atgttggcac tgatgagggt gtcagtgaag tgcttcatgt ggcaggagaa
     7201 aaaaggctgc accggtgcgt cagcagaata tgtgatacag gatatattcc gcttcctcgc
     7261 tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg
     7321 gcggagattt cctggaagat gccaggaaga tacttaacag ggaagtgaga gggccgcggc
     7381 aaagccgttt ttccataggc tccgcccccc tgacaagcat cacgaaatct gacgctcaaa
     7441 tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggcggctc
     7501 cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt gtcattccgc tgttatggcc
     7561 gcgtttgtct cattccacgc ctgacactca gttcc
//
