LOCUS       sequence-370307-        7415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cas12b 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
FEATURES             Location/Qualifiers
     source          1..7415
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     970..989
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1009..1086)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1132..1150
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1151..1175
                     /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             1231..1248
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        4767..4785
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4786..4810
                     /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             4919..4963
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5001..5019)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5015..5062
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5283..5942)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5857..5876
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5943..6045)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(6437..6454)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6571..7116)
                     /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(6651..6670)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7341..7415,1..1008))
                     /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"
ORIGIN
        1 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
       61 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      181 ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat
      241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
      301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
      361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
      421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
      481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      601 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
      661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     1081 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaaa ttaatacgac
     1141 tcactatagg ggaattgtga gcggataaca attcccctgt agaaataatt ttgtttaact
     1201 ttaataagga gatataccat gggcagcagc catcaccatc atcaccacag ccaggatcct
     1261 taggagggcg ctagatggcc gtcaaatcca tcaaagtgaa acttcgtctc gacgatatgc
     1321 cggagattcg ggccggtcta tggaaacttc ataaggaagt caatgcgggg gttcgatatt
     1381 acacggaatg gctcagtctt ctccgtcaag agaacttgta tcgaagaagt ccgaatgggg
     1441 acggagagca agaatgtgat aagactgcag aagaatgcaa agccgaattg ttggagcggc
     1501 tgcgcgcgcg tcaagtggag aatggacacc gtggtccggc gggatcggac gatgaattgc
     1561 tgcagttggc gcgtcaactc tatgagttgt tggttccgca ggcgataggt gcgaaaggcg
     1621 acgcgcagca aattgcccgc aaatttttga gccccttggc cgacaaggac gcagttggtg
     1681 ggcttggaat cgcgaaggcg gggaacaaac cgcggtgggt tcgcatgcgc gaagcggggg
     1741 aaccaggctg ggaagaggag aaggagaagg ctgagacgag gaaatctgcg gatcggactg
     1801 cggatgtttt gcgcgcgctc gcggattttg ggttaaagcc actgatgcgc gtatacaccg
     1861 attctgagat gtcatcggtg gagtggaaac cgcttcggaa gggacaagcc gttcggacgt
     1921 gggataggga catgttccaa caagctatcg aacggatgat gtcgtgggag tcgtggaatc
     1981 agcgcgttgg gcaagagtac gcgaaactcg tagaacaaaa aaatcgattt gagcagaaga
     2041 atttcgtcgg ccaggaacat ctggtccatc tcgtcaatca gttgcaacaa gatatgaaag
     2101 aagcatcgcc cggactcgaa tcgaaagagc aaaccgcgca ctatgtgacg ggacgggcat
     2161 tgcgcggatc ggacaaggta tttgagaagt gggggaaact cgcccccgat gcacctttcg
     2221 atttgtacga cgccgaaatc aagaatgtgc agagacgtaa cacgagacga ttcggatcac
     2281 atgacttgtt cgcaaaattg gcagagccag agtatcaggc cctgtggcgc gaagatgctt
     2341 cgtttctcac gcgttacgcg gtgtacaaca gcatccttcg caaactgaat cacgccaaaa
     2401 tgttcgcgac gtttactttg ccggatgcaa cggcgcaccc gatttggact cgcttcgata
     2461 aattgggtgg gaatttgcac cagtacacct ttttgttcaa cgaatttgga gaacgcaggc
     2521 acgcgattcg ttttcacaag ctattgaaag tcgagaatgg tgtcgcaaga gaagttgatg
     2581 atgtcaccgt gcccatttca atgtcagagc aattggataa tctgcttccc agagatccca
     2641 atgaaccgat tgcgctatat tttcgagatt acggagccga acagcatttc acaggtgaat
     2701 ttggtggcgc gaagatccag tgccgccggg atcagctggc tcatatgcac cgacgcagag
     2761 gggcgaggga tgtttatctc aatgtcagcg tacgtgtgca gagtcagtct gaggcgcggg
     2821 gagaacgtcg cccgccgtat gcggcagtat ttcgtctggt cggggacaac catcgcgcgt
     2881 ttgtccattt cgataaacta tcggattatc ttgcggaaca tccggatgat gggaagctcg
     2941 ggtcggaggg gttgctttcc gggctgcggg tgatgagtgt cgatctcggc cttcgcacat
     3001 ctgcatcgat ttccgttttt cgcgttgccc ggaaggacga gttgaagccg aactcaaaag
     3061 gtcgtgtacc gtttttcttt ccgataaaag ggaatgacaa tctcgtcgcg gttcatgagc
     3121 gatcacaact cttgaagctg cctggcgaaa cggagtcgaa ggacctgcgt gctatccgag
     3181 aagaacgcca acggacattg cggcagttgc ggacgcaact ggcgtatttg cggctgctcg
     3241 tgcggtgtgg gtcggaagat gtggggcggc gtgaacggag ttgggcaaag cttatcgagc
     3301 agccggtgga tgcggccaat cacatgacac cggattggcg cgaggctttt gaaaacgaac
     3361 ttcagaagct taagtcactc catggtatct gtagcgacaa ggaatggatg gatgctgtct
     3421 acgagagcgt tcgccgcgtg tggcgtcaca tgggcaaaca ggttcgcgat tggcgaaagg
     3481 acgtacgaag cggagagcgg cccaagattc gcggctatgc gaaagacgtg gtcggtggaa
     3541 actcgattga gcaaatcgag tatctggaac gtcagtacaa gttcctcaag agttggagct
     3601 tctttggtaa ggtgtcggga caagtgattc gtgcggagaa gggatctcgt tttgcgatca
     3661 cgctgcgcga acacattgat cacgcgaagg aagatcggct gaagaaattg gcggatcgca
     3721 tcattatgga ggctctcggc tatgtgtacg cgttggatga gcgcggcaaa ggaaagtggg
     3781 ttgcgaagta tccgccgtgc cagctcatcc tgctggagga attgagcgag taccagttca
     3841 ataacgacag gcctccgagc gaaaacaacc agttgatgca atggagtcat cgcggcgtgt
     3901 tccaggagtt gataaatcag gcccaagtcc atgatttact cgttgggacg atgtatgcag
     3961 cgttctcgtc gcgattcgac gcgcgaactg gggcaccggg tatccgctgt cgccgggttc
     4021 cggcgcgttg cacccaggag cacaatccag aaccatttcc ttggtggctg aacaagtttg
     4081 tggtggaaca tacgttggat gcttgtcccc tacgcgcaga cgacctcatc ccaacgggtg
     4141 aaggagagat ttttgtctcg ccgttcagcg cggaggaggg ggactttcat cagattcacg
     4201 ccgacctgaa tgcggcgcaa aatctgcagc agcgactctg gtctgatttt gatatcagtc
     4261 aaattcggtt gcggtgtgat tggggtgaag tggacggtga actcgttctg atcccaaggc
     4321 ttacaggaaa acgaacggcg gattcatata gcaacaaggt gttttatacc aatacaggtg
     4381 tcacctatta tgagcgagag cgggggaaga agcggagaaa ggttttcgcg caagagaaat
     4441 tgtcggagga agaggcggag ttgctcgtgg aagcagacga ggcgagggag aaatcggtcg
     4501 ttttgatgcg tgatccgtct ggcatcatca atcggggaaa ttggaccagg caaaaggaat
     4561 tttggtcgat ggtgaaccag cggatcgaag gatacttggt caagcagatt cgctcgcgcg
     4621 ttccattaca agatagtgcg tgtgaaaaca cgggggatat ttaagaattc gagctcggcg
     4681 cgcctgcagg tcgacaagct tgcggccgca taatgcttaa gtcgaacaga aagtaatcgt
     4741 attgtacacg gccgcataat cgaaattaat acgactcact ataggggaat tgtgagcgga
     4801 taacaattcc ccatcttagt atattagtta agtataagaa ggagatatac atatggcaga
     4861 tctcaattgg atatcggccg gccacgcgat cgctgacgtc ggtaccctcg agtctggtaa
     4921 agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta
     4981 attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     5041 acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact
     5101 gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc
     5161 ctgccctgaa ccgacgaccg ggtcgaattt gctttcgaat ttctgccatt catccgctta
     5221 ttatcactta ttcaggcgta gcaaccaggc gtttaagggc accaataact gccttaaaaa
     5281 aattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg
     5341 acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg
     5401 tcgccttgcg tataatattt gcccatagtg aaaacggggg cgaagaagtt gtccatattg
     5461 gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata
     5521 ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc
     5581 gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac
     5641 gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc
     5701 tcaccgtctt tcattgccat acggaactcc ggatgagcat tcatcaggcg ggcaagaatg
     5761 tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta
     5821 atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa
     5881 tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc
     5941 attttagctt ccttagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat
     6001 cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc
     6061 caaaagttgg cccagggctt cccggtatca acagggacac caggatttat ttattctgcg
     6121 aagtgatctt ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac
     6181 ttactgattt agtgtatgat ggtgtttttg aggtgctcca gtggcttctg tttctatcag
     6241 ctgtccctcc tgttcagcta ctgacggggt ggtgcgtaac ggcaaaagca ccgccggaca
     6301 tcagcgctag cggagtgtat actggcttac tatgttggca ctgatgaggg tgtcagtgaa
     6361 gtgcttcatg tggcaggaga aaaaaggctg caccggtgcg tcagcagaat atgtgataca
     6421 ggatatattc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag
     6481 cggaaatggc ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca
     6541 gggaagtgag agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca
     6601 tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca
     6661 ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg
     6721 tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt
     6781 aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg
     6841 ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg
     6901 cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc
     6961 taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa
     7021 gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca
     7081 gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata
     7141 aaatatttct agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc
     7201 atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg aaggagctga
     7261 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     7321 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     7381 tgcattaatg aatcggccaa cgcgcgggga gaggc
//
