LOCUS       sequence-370316-        7595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes inactive Cas12a containing mutation in the PI domain (K613A,
            K617A) 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: pCas12(PI)
FEATURES             Location/Qualifiers
     source          1..7595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(527..1609)
                     /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     1571..1590
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1610..1687)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1733..1752
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1733..1751
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1752..1776
                     /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(5782..5800)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5796..5843
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(6064..6723)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6638..6657
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6724..6826)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(7218..7235)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(7352..7595,1..302))
                     /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(7432..7451)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
       61 ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta gaggagttag
      121 tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt gactgcgctc
      181 ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc
      241 cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca aaacgatctc
      301 aagaagatca tcttattaat cagataaaat atttctagat ttcagtgcaa tttatctctt
      361 caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat
      421 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
      481 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
      541 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
      601 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
      661 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
      721 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
      781 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
      841 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
      901 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
      961 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     1021 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     1081 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     1141 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     1201 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     1261 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     1321 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     1381 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     1441 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     1501 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     1561 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     1621 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     1681 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa attaatacga
     1741 ctcactatag gggaattgtg agcggataac aattcccctg tagaaataat tttgtttaac
     1801 tttaataagg agatatacca tggatgtcaa tttatcaaga atttgttaat aaatatagtt
     1861 taagtaaaac tctaagattt gagttaatcc cacagggtaa aacacttgaa aacataaaag
     1921 caagaggttt gattttagat gatgagaaaa gagctaaaga ctacaaaaag gctaaacaaa
     1981 taattgataa atatcatcag ttttttatag aggagatatt aagttcggtt tgtattagcg
     2041 aagatttatt acaaaactat tctgatgttt attttaaact taaaaagagt gatgatgata
     2101 atctacaaaa agattttaaa agtgcaaaag atacgataaa gaaacaaata tctgaatata
     2161 taaaggactc agagaaattt aagaatttgt ttaatcaaaa ccttatcgat gctaaaaaag
     2221 ggcaagagtc agatttaatt ctatggctaa agcaatctaa ggataatggt atagaactat
     2281 ttaaagccaa tagtgatatc acagatatag atgaggcgtt agaaataatc aaatctttta
     2341 aaggttggac aacttatttt aagggttttc atgaaaatag aaaaaatgtt tatagtagca
     2401 atgatattcc tacatctatt atttatagga tagtagatga taatttgcct aaatttctag
     2461 aaaataaagc taagtatgag agtttaaaag acaaagctcc agaagctata aactatgaac
     2521 aaattaaaaa agatttggca gaagagctaa cctttgatat tgactacaaa acatctgaag
     2581 ttaatcaaag agttttttca cttgatgaag tttttgagat agcaaacttt aataattatc
     2641 taaatcaaag tggtattact aaatttaata ctattattgg tggtaaattt gtaaatggtg
     2701 aaaatacaaa gagaaaaggt ataaatgaat atataaatct atactcacag caaataaatg
     2761 ataaaacact caaaaaatat aaaatgagtg ttttatttaa gcaaatttta agtgatacag
     2821 aatctaaatc ttttgtaatt gataagttag aagatgatag tgatgtagtt acaacgatgc
     2881 aaagttttta tgagcaaata gcagctttta aaacagtaga agaaaaatct attaaagaaa
     2941 cactatcttt attatttgat gatttaaaag ctcaaaaact tgatttgagt aaaatttatt
     3001 ttaaaaatga taaatctctt actgatctat cacaacaagt ttttgatgat tatagtgtta
     3061 ttggtacagc ggtactagaa tatataactc aacaaatagc acctaaaaat cttgataacc
     3121 ctagtaagaa agagcaagaa ttaatagcca aaaaaactga aaaagcaaaa tacttatctc
     3181 tagaaactat aaagcttgcc ttagaagaat ttaataagca tagagatata gataaacagt
     3241 gtaggtttga agaaatactt gcaaactttg cggctattcc gatgatattt gatgaaatag
     3301 ctcaaaacaa agacaatttg gcacagatat ctatcaaata tcaaaatcaa ggtaaaaaag
     3361 acctacttca agctagtgcg gaagatgatg ttaaagctat caaggatctt ttagatcaaa
     3421 ctaataatct cttacataaa ctaaaaatat ttcatattag tcagtcagaa gataaggcaa
     3481 atattttaga caaggatgag catttttatc tagtatttga ggagtgctac tttgagctag
     3541 cgaatatagt gcctctttat aacaaaatta gaaactatat aactcaaaag ccatatagtg
     3601 atgagaaatt taagctcaat tttgagaact cgactttggc taatggttgg gataaaaatg
     3661 cagagcctga caatacggca attttattta tcaaagatga taaatattat ctgggtgtga
     3721 tgaataagaa aaataacaaa atatttgatg ataaagctat caaagaaaat aaaggcgagg
     3781 gttataaaaa aattgtttat aaacttttac ctggcgcaaa taaaatgtta cctgcggttt
     3841 tcttttctgc taaatctata aaattttata atcctagtga agatatactt agaataagaa
     3901 atcattccac acatacaaaa aatggtagtc ctcaaaaagg atatgaaaaa tttgagttta
     3961 atattgaaga ttgccgaaaa tttatagatt tttataaaca gtctataagt aagcatccgg
     4021 agtggaaaga ttttggattt agattttctg atactcaaag atataattct atagatgaat
     4081 tttatagaga agttgaaaat caaggctaca aactaacttt tgaaaatata tcagagagct
     4141 atattgatag cgtagttaat cagggtaaat tgtacctatt ccaaatctat aataaagatt
     4201 tttcagctta tagcaaaggg cgaccaaatc tacatacttt atattggaaa gcgctgtttg
     4261 atgagagaaa tcttcaagat gtggtttata agctaaatgg tgaggcagag cttttttatc
     4321 gtaaacaatc aatacctaaa aaaatcactc acccagctaa agaggcaata gctaataaaa
     4381 acaaagataa tcctaaaaaa gagagtgttt ttgaatatga tttaatcaaa gataaacgct
     4441 ttactgaaga taagtttttc tttcactgtc ctattacaat caattttaaa tctagtggag
     4501 ctaataagtt taatgatgaa atcaatttat tgctaaaaga aaaagcaaat gatgttcata
     4561 tattaagtat agatagaggt gaaagacatt tagcttacta tactttggta gatggtaaag
     4621 gcaatatcat caaacaagat actttcaaca tcattggtaa tgatagaatg aaaacaaact
     4681 accatgataa gcttgctgca atagagaaag atagggattc agctaggaaa gactggaaaa
     4741 agataaataa catcaaagag atgaaagagg gctatctatc tcaggtagtt catgaaatag
     4801 ctaagctagt tatagagtat aatgctattg tggtttttga ggatttaaat tttggattta
     4861 aaagagggcg tttcaaggta gagaagcagg tctatcaaaa gttagaaaaa atgctaattg
     4921 agaaactaaa ctatctagtt ttcaaagata atgagtttga taaaactggg ggagtgctta
     4981 gagcttatca gctaacagca ccttttgaga cttttaaaaa gatgggtaaa caaacaggta
     5041 ttatctacta tgtaccagct ggttttactt caaaaatttg tcctgtaact ggttttgtaa
     5101 atcagttata tcctaagtat gaaagtgtca gcaaatctca agagttcttt agtaagtttg
     5161 acaagatttg ttataacctt gataagggct attttgagtt tagttttgat tataaaaact
     5221 ttggtgacaa ggctgccaaa ggcaagtgga ctatagctag ctttgggagt agattgatta
     5281 actttagaaa ttcagataaa aatcataatt gggatactcg agaagtttat ccaactaaag
     5341 agttggagaa attgctaaaa gattattcta tcgaatatgg gcatggcgaa tgtatcaaag
     5401 cagctatttg cggtgagagc gacaaaaagt tttttgctaa gctaactagt gtcctaaata
     5461 ctatcttaca aatgcgtaac tcaaaaacag gtactgagtt agattatcta atttcaccag
     5521 tagcagatgt aaatggcaat ttctttgatt cgcgacaggc gccaaaaaat atgcctcaag
     5581 atgctgatgc caatggtgct tatcatattg ggctaaaagg tctgatgcta ctaggtagga
     5641 tcaaaaataa tcaagagggc aaaaaactca atttggttat caaaaatgaa gagtattttg
     5701 agttcgtgca gaataggaat aactaaccca gagaattcga gctcggcgcg cctgcaggtc
     5761 gacaagcttg cgctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     5821 aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac acggtcacac
     5881 tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac
     5941 cctgccctga accgacgacc gggtcgaatt tgctttcgaa tttctgccat tcatccgctt
     6001 attatcactt attcaggcgt agcaaccagg cgtttaaggg caccaataac tgccttaaaa
     6061 aaattacgcc ccgccctgcc actcatcgca gtactgttgt aattcattaa gcattctgcc
     6121 gacatggaag ccatcacaaa cggcatgatg aacctgaatc gccagcggca tcagcacctt
     6181 gtcgccttgc gtataatatt tgcccatagt gaaaacgggg gcgaagaagt tgtccatatt
     6241 ggccacgttt aaatcaaaac tggtgaaact cacccaggga ttggctgaga cgaaaaacat
     6301 attctcaata aaccctttag ggaaataggc caggttttca ccgtaacacg ccacatcttg
     6361 cgaatatatg tgtagaaact gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa
     6421 cgtttcagtt tgctcatgga aaacggtgta acaagggtga acactatccc atatcaccag
     6481 ctcaccgtct ttcattgcca tacggaactc cggatgagca ttcatcaggc gggcaagaat
     6541 gtgaataaag gccggataaa acttgtgctt atttttcttt acggtcttta aaaaggccgt
     6601 aatatccagc tgaacggtct ggttataggt acattgagca actgactgaa atgcctcaaa
     6661 atgttcttta cgatgccatt gggatatatc aacggtggta tatccagtga tttttttctc
     6721 cattttagct tccttagctc ctgaaaatct cgataactca aaaaatacgc ccggtagtga
     6781 tcttatttca ttatggtgaa agttggaacc tcttacgtgc cgatcaacgt ctcattttcg
     6841 ccaaaagttg gcccagggct tcccggtatc aacagggaca ccaggattta tttattctgc
     6901 gaagtgatct tccgtcacag gtatttattc ggcgcaaagt gcgtcgggtg atgctgccaa
     6961 cttactgatt tagtgtatga tggtgttttt gaggtgctcc agtggcttct gtttctatca
     7021 gctgtccctc ctgttcagct actgacgggg tggtgcgtaa cggcaaaagc accgccggac
     7081 atcagcgcta gcggagtgta tactggctta ctatgttggc actgatgagg gtgtcagtga
     7141 agtgcttcat gtggcaggag aaaaaaggct gcaccggtgc gtcagcagaa tatgtgatac
     7201 aggatatatt ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga
     7261 gcggaaatgg cttacgaacg gggcggagat ttcctggaag atgccaggaa gatacttaac
     7321 agggaagtga gagggccgcg gcaaagccgt ttttccatag gctccgcccc cctgacaagc
     7381 atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc
     7441 aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg
     7501 gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg
     7561 taggcagttc gctccaagct ggactgtatg cacga
//
