LOCUS       sequence-370298-        7415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes inactive Cas12b containing mutation in the PI domain (R122A,
            G143P) from A. acidoterrestris (type V-B).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7415)
  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 7415)
  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: pCas12b(PI)
FEATURES             Location/Qualifiers
     source          1..7415
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(553..1635)
                     /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     1597..1616
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1636..1713)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1759..1777
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1778..1802
                     /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             1858..1875
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        5394..5412
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5413..5437
                     /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             5546..5590
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5628..5646)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5642..5689
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5910..6569)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6484..6503
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6570..6672)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(7064..7081)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(7198..7415,1..328))
                     /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(7278..7297)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgctccaagc tggactgtat gcacgaaccc cccgttcagt ccgaccgctg cgccttatcc
       61 ggtaactatc gtcttgagtc caacccggaa agacatgcaa aagcaccact ggcagcagcc
      121 actggtaatt gatttagagg agttagtctt gaagtcatgc gccggttaag gctaaactga
      181 aaggacaagt tttggtgact gcgctcctcc aagccagtta cctcggttca aagagttggt
      241 agctcagaga accttcgaaa aaccgccctg caaggcggtt ttttcgtttt cagagcaaga
      301 gattacgcgc agaccaaaac gatctcaaga agatcatctt attaatcaga taaaatattt
      361 ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata
      421 taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc ggaaggagct gactgggttg
      481 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
      541 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
      601 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
      661 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
      721 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
      781 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc
      841 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
      901 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
      961 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     1021 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     1081 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     1141 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     1201 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     1261 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     1321 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     1381 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     1441 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     1501 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     1561 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     1621 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     1681 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     1741 actcctgcat taggaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     1801 cccctgtaga aataattttg tttaacttta ataaggagat ataccatggg cagcagccat
     1861 caccatcatc accacagcca ggatccttag gagggcgcta gatggccgtc aaatccatca
     1921 aagtgaaact tcgtctcgac gatatgccgg agattcgggc cggtctatgg aaacttcata
     1981 aggaagtcaa tgcgggggtt cgatattaca cggaatggct cagtcttctc cgtcaagaga
     2041 acttgtatcg aagaagtccg aatggggacg gagagcaaga atgtgataag actgcagaag
     2101 aatgcaaagc cgaattgttg gagcggctgc gcgcgcgtca agtggagaat ggacaccgtg
     2161 gtccggcggg atcggacgat gaattgctgc agttggcgcg tcaactctat gagttgttgg
     2221 ttccgcaggc gataggtgcg aaaggcgacg cgcagcaaat tgccgccaaa tttttgagcc
     2281 ccttggccga caaggacgca gttggtgggc ttggaatcgc gaaggcgccg aacaaaccgc
     2341 ggtgggttcg catgcgcgaa gcgggggaac caggctggga agaggagaag gagaaggctg
     2401 agacgaggaa atctgcggat cggactgcgg atgttttgcg cgcgctcgcg gattttgggt
     2461 taaagccact gatgcgcgta tacaccgatt ctgagatgtc atcggtggag tggaaaccgc
     2521 ttcggaaggg acaagccgtt cggacgtggg atagggacat gttccaacaa gctatcgaac
     2581 ggatgatgtc gtgggagtcg tggaatcagc gcgttgggca agagtacgcg aaactcgtag
     2641 aacaaaaaaa tcgatttgag cagaagaatt tcgtcggcca ggaacatctg gtccatctcg
     2701 tcaatcagtt gcaacaagat atgaaagaag catcgcccgg actcgaatcg aaagagcaaa
     2761 ccgcgcacta tgtgacggga cgggcattgc gcggatcgga caaggtattt gagaagtggg
     2821 ggaaactcgc ccccgatgca cctttcgatt tgtacgacgc cgaaatcaag aatgtgcaga
     2881 gacgtaacac gagacgattc ggatcacatg acttgttcgc aaaattggca gagccagagt
     2941 atcaggccct gtggcgcgaa gatgcttcgt ttctcacgcg ttacgcggtg tacaacagca
     3001 tccttcgcaa actgaatcac gccaaaatgt tcgcgacgtt tactttgccg gatgcaacgg
     3061 cgcacccgat ttggactcgc ttcgataaat tgggtgggaa tttgcaccag tacacctttt
     3121 tgttcaacga atttggagaa cgcaggcacg cgattcgttt tcacaagcta ttgaaagtcg
     3181 agaatggtgt cgcaagagaa gttgatgatg tcaccgtgcc catttcaatg tcagagcaat
     3241 tggataatct gcttcccaga gatcccaatg aaccgattgc gctatatttt cgagattacg
     3301 gagccgaaca gcatttcaca ggtgaatttg gtggcgcgaa gatccagtgc cgccgggatc
     3361 agctggctca tatgcaccga cgcagagggg cgagggatgt ttatctcaat gtcagcgtac
     3421 gtgtgcagag tcagtctgag gcgcggggag aacgtcgccc gccgtatgcg gcagtatttc
     3481 gtctggtcgg ggacaaccat cgcgcgtttg tccatttcga taaactatcg gattatcttg
     3541 cggaacatcc ggatgatggg aagctcgggt cggaggggtt gctttccggg ctgcgggtga
     3601 tgagtgtcga tctcggcctt cgcacatctg catcgatttc cgtttttcgc gttgcccgga
     3661 aggacgagtt gaagccgaac tcaaaaggtc gtgtaccgtt tttctttccg ataaaaggga
     3721 atgacaatct cgtcgcggtt catgagcgat cacaactctt gaagctgcct ggcgaaacgg
     3781 agtcgaagga cctgcgtgct atccgagaag aacgccaacg gacattgcgg cagttgcgga
     3841 cgcaactggc gtatttgcgg ctgctcgtgc ggtgtgggtc ggaagatgtg gggcggcgtg
     3901 aacggagttg ggcaaagctt atcgagcagc cggtggatgc ggccaatcac atgacaccgg
     3961 attggcgcga ggcttttgaa aacgaacttc agaagcttaa gtcactccat ggtatctgta
     4021 gcgacaagga atggatggat gctgtctacg agagcgttcg ccgcgtgtgg cgtcacatgg
     4081 gcaaacaggt tcgcgattgg cgaaaggacg tacgaagcgg agagcggccc aagattcgcg
     4141 gctatgcgaa agacgtggtc ggtggaaact cgattgagca aatcgagtat ctggaacgtc
     4201 agtacaagtt cctcaagagt tggagcttct ttggtaaggt gtcgggacaa gtgattcgtg
     4261 cggagaaggg atctcgtttt gcgatcacgc tgcgcgaaca cattgatcac gcgaaggaag
     4321 atcggctgaa gaaattggcg gatcgcatca ttatggaggc tctcggctat gtgtacgcgt
     4381 tggatgagcg cggcaaagga aagtgggttg cgaagtatcc gccgtgccag ctcatcctgc
     4441 tggaggaatt gagcgagtac cagttcaata acgacaggcc tccgagcgaa aacaaccagt
     4501 tgatgcaatg gagtcatcgc ggcgtgttcc aggagttgat aaatcaggcc caagtccatg
     4561 atttactcgt tgggacgatg tatgcagcgt tctcgtcgcg attcgacgcg cgaactgggg
     4621 caccgggtat ccgctgtcgc cgggttccgg cgcgttgcac ccaggagcac aatccagaac
     4681 catttccttg gtggctgaac aagtttgtgg tggaacatac gttggatgct tgtcccctac
     4741 gcgcagacga cctcatccca acgggtgaag gagagatttt tgtctcgccg ttcagcgcgg
     4801 aggaggggga ctttcatcag attcacgccg acctgaatgc ggcgcaaaat ctgcagcagc
     4861 gactctggtc tgattttgat atcagtcaaa ttcggttgcg gtgtgattgg ggtgaagtgg
     4921 acggtgaact cgttctgatc ccaaggctta caggaaaacg aacggcggat tcatatagca
     4981 acaaggtgtt ttataccaat acaggtgtca cctattatga gcgagagcgg gggaagaagc
     5041 ggagaaaggt tttcgcgcaa gagaaattgt cggaggaaga ggcggagttg ctcgtggaag
     5101 cagacgaggc gagggagaaa tcggtcgttt tgatgcgtga tccgtctggc atcatcaatc
     5161 ggggaaattg gaccaggcaa aaggaatttt ggtcgatggt gaaccagcgg atcgaaggat
     5221 acttggtcaa gcagattcgc tcgcgcgttc cattacaaga tagtgcgtgt gaaaacacgg
     5281 gggatattta agaattcgag ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa
     5341 tgcttaagtc gaacagaaag taatcgtatt gtacacggcc gcataatcga aattaatacg
     5401 actcactata ggggaattgt gagcggataa caattcccca tcttagtata ttagttaagt
     5461 ataagaagga gatatacata tggcagatct caattggata tcggccggcc acgcgatcgc
     5521 tgacgtcggt accctcgagt ctggtaaaga aaccgctgct gcgaaatttg aacgccagca
     5581 catggactcg tctactagcg cagcttaatt aacctaggct gctgccaccg ctgagcaata
     5641 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaacctca
     5701 ggcatttgag aagcacacgg tcacactgct tccggtagtc aataaaccgg taaaccagca
     5761 atagacataa gcggctattt aacgaccctg ccctgaaccg acgaccgggt cgaatttgct
     5821 ttcgaatttc tgccattcat ccgcttatta tcacttattc aggcgtagca accaggcgtt
     5881 taagggcacc aataactgcc ttaaaaaaat tacgccccgc cctgccactc atcgcagtac
     5941 tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc
     6001 tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catagtgaaa
     6061 acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc
     6121 cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg
     6181 ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg
     6241 tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa
     6301 gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg gaactccgga
     6361 tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt
     6421 ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat
     6481 tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg
     6541 gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgat
     6601 aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt
     6661 acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc ggtatcaaca
     6721 gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat ttattcggcg
     6781 caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt gtttttgagg
     6841 tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg acggggtggt
     6901 gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg agtgtatact ggcttactat
     6961 gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa aaggctgcac
     7021 cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc actgactcgc
     7081 tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc ggagatttcc
     7141 tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa agccgttttt
     7201 ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc agtggtggcg
     7261 aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc tcgtgcgctc
     7321 tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc gtttgtctca
     7381 ttccacgcct gacactcagt tccgggtagg cagtt
//
