LOCUS       sequence-370306-        7415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes catalytically inactive Cas12b (E848A, D977A) 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: pdCas12b(RuvC)
FEATURES             Location/Qualifiers
     source          1..7415
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(580..1662)
                     /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     1624..1643
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1663..1740)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1786..1804
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1805..1829
                     /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             1885..1902
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        5421..5439
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5440..5464
                     /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             5573..5617
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5655..5673)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5669..5716
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5937..6596)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6511..6530
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6597..6699)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(7091..7108)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(7225..7415,1..355))
                     /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(7305..7324)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgacactca gttccgggta ggcagttcgc tccaagctgg actgtatgca cgaacccccc
       61 gttcagtccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggaaaga
      121 catgcaaaag caccactggc agcagccact ggtaattgat ttagaggagt tagtcttgaa
      181 gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt ggtgactgcg ctcctccaag
      241 ccagttacct cggttcaaag agttggtagc tcagagaacc ttcgaaaaac cgccctgcaa
      301 ggcggttttt tcgttttcag agcaagagat tacgcgcaga ccaaaacgat ctcaagaaga
      361 tcatcttatt aatcagataa aatatttcta gatttcagtg caatttatct cttcaaatgt
      421 agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc
      481 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
      541 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
      601 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
      661 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
      721 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
      781 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
      841 gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg
      901 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
      961 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     1021 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     1081 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     1141 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     1201 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     1261 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     1321 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     1381 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     1441 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     1501 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     1561 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     1621 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     1681 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     1741 cgggatctcg acgctctccc ttatgcgact cctgcattag gaaattaata cgactcacta
     1801 taggggaatt gtgagcggat aacaattccc ctgtagaaat aattttgttt aactttaata
     1861 aggagatata ccatgggcag cagccatcac catcatcacc acagccagga tccttaggag
     1921 ggcgctagat ggccgtcaaa tccatcaaag tgaaacttcg tctcgacgat atgccggaga
     1981 ttcgggccgg tctatggaaa cttcataagg aagtcaatgc gggggttcga tattacacgg
     2041 aatggctcag tcttctccgt caagagaact tgtatcgaag aagtccgaat ggggacggag
     2101 agcaagaatg tgataagact gcagaagaat gcaaagccga attgttggag cggctgcgcg
     2161 cgcgtcaagt ggagaatgga caccgtggtc cggcgggatc ggacgatgaa ttgctgcagt
     2221 tggcgcgtca actctatgag ttgttggttc cgcaggcgat aggtgcgaaa ggcgacgcgc
     2281 agcaaattgc ccgcaaattt ttgagcccct tggccgacaa ggacgcagtt ggtgggcttg
     2341 gaatcgcgaa ggcggggaac aaaccgcggt gggttcgcat gcgcgaagcg ggggaaccag
     2401 gctgggaaga ggagaaggag aaggctgaga cgaggaaatc tgcggatcgg actgcggatg
     2461 ttttgcgcgc gctcgcggat tttgggttaa agccactgat gcgcgtatac accgattctg
     2521 agatgtcatc ggtggagtgg aaaccgcttc ggaagggaca agccgttcgg acgtgggata
     2581 gggacatgtt ccaacaagct atcgaacgga tgatgtcgtg ggagtcgtgg aatcagcgcg
     2641 ttgggcaaga gtacgcgaaa ctcgtagaac aaaaaaatcg atttgagcag aagaatttcg
     2701 tcggccagga acatctggtc catctcgtca atcagttgca acaagatatg aaagaagcat
     2761 cgcccggact cgaatcgaaa gagcaaaccg cgcactatgt gacgggacgg gcattgcgcg
     2821 gatcggacaa ggtatttgag aagtggggga aactcgcccc cgatgcacct ttcgatttgt
     2881 acgacgccga aatcaagaat gtgcagagac gtaacacgag acgattcgga tcacatgact
     2941 tgttcgcaaa attggcagag ccagagtatc aggccctgtg gcgcgaagat gcttcgtttc
     3001 tcacgcgtta cgcggtgtac aacagcatcc ttcgcaaact gaatcacgcc aaaatgttcg
     3061 cgacgtttac tttgccggat gcaacggcgc acccgatttg gactcgcttc gataaattgg
     3121 gtgggaattt gcaccagtac acctttttgt tcaacgaatt tggagaacgc aggcacgcga
     3181 ttcgttttca caagctattg aaagtcgaga atggtgtcgc aagagaagtt gatgatgtca
     3241 ccgtgcccat ttcaatgtca gagcaattgg ataatctgct tcccagagat cccaatgaac
     3301 cgattgcgct atattttcga gattacggag ccgaacagca tttcacaggt gaatttggtg
     3361 gcgcgaagat ccagtgccgc cgggatcagc tggctcatat gcaccgacgc agaggggcga
     3421 gggatgttta tctcaatgtc agcgtacgtg tgcagagtca gtctgaggcg cggggagaac
     3481 gtcgcccgcc gtatgcggca gtatttcgtc tggtcgggga caaccatcgc gcgtttgtcc
     3541 atttcgataa actatcggat tatcttgcgg aacatccgga tgatgggaag ctcgggtcgg
     3601 aggggttgct ttccgggctg cgggtgatga gtgtcgatct cggccttcgc acatctgcat
     3661 cgatttccgt ttttcgcgtt gcccggaagg acgagttgaa gccgaactca aaaggtcgtg
     3721 taccgttttt ctttccgata aaagggaatg acaatctcgt cgcggttcat gagcgatcac
     3781 aactcttgaa gctgcctggc gaaacggagt cgaaggacct gcgtgctatc cgagaagaac
     3841 gccaacggac attgcggcag ttgcggacgc aactggcgta tttgcggctg ctcgtgcggt
     3901 gtgggtcgga agatgtgggg cggcgtgaac ggagttgggc aaagcttatc gagcagccgg
     3961 tggatgcggc caatcacatg acaccggatt ggcgcgaggc ttttgaaaac gaacttcaga
     4021 agcttaagtc actccatggt atctgtagcg acaaggaatg gatggatgct gtctacgaga
     4081 gcgttcgccg cgtgtggcgt cacatgggca aacaggttcg cgattggcga aaggacgtac
     4141 gaagcggaga gcggcccaag attcgcggct atgcgaaaga cgtggtcggt ggaaactcga
     4201 ttgagcaaat cgagtatctg gaacgtcagt acaagttcct caagagttgg agcttctttg
     4261 gtaaggtgtc gggacaagtg attcgtgcgg agaagggatc tcgttttgcg atcacgctgc
     4321 gcgaacacat tgatcacgcg aaggaagatc ggctgaagaa attggcggat cgcatcatta
     4381 tggaggctct cggctatgtg tacgcgttgg atgagcgcgg caaaggaaag tgggttgcga
     4441 agtatccgcc gtgccagctc atcctgctgg ccgaattgag cgagtaccag ttcaataacg
     4501 acaggcctcc gagcgaaaac aaccagttga tgcaatggag tcatcgcggc gtgttccagg
     4561 agttgataaa tcaggcccaa gtccatgatt tactcgttgg gacgatgtat gcagcgttct
     4621 cgtcgcgatt cgacgcgcga actggggcac cgggtatccg ctgtcgccgg gttccggcgc
     4681 gttgcaccca ggagcacaat ccagaaccat ttccttggtg gctgaacaag tttgtggtgg
     4741 aacatacgtt ggatgcttgt cccctacgcg cagacgacct catcccaacg ggtgaaggag
     4801 agatttttgt ctcgccgttc agcgcggagg agggggactt tcatcagatt cacgccgccc
     4861 tgaatgcggc gcaaaatctg cagcagcgac tctggtctga ttttgatatc agtcaaattc
     4921 ggttgcggtg tgattggggt gaagtggacg gtgaactcgt tctgatccca aggcttacag
     4981 gaaaacgaac ggcggattca tatagcaaca aggtgtttta taccaataca ggtgtcacct
     5041 attatgagcg agagcggggg aagaagcgga gaaaggtttt cgcgcaagag aaattgtcgg
     5101 aggaagaggc ggagttgctc gtggaagcag acgaggcgag ggagaaatcg gtcgttttga
     5161 tgcgtgatcc gtctggcatc atcaatcggg gaaattggac caggcaaaag gaattttggt
     5221 cgatggtgaa ccagcggatc gaaggatact tggtcaagca gattcgctcg cgcgttccat
     5281 tacaagatag tgcgtgtgaa aacacggggg atatttaaga attcgagctc ggcgcgcctg
     5341 caggtcgaca agcttgcggc cgcataatgc ttaagtcgaa cagaaagtaa tcgtattgta
     5401 cacggccgca taatcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     5461 ttccccatct tagtatatta gttaagtata agaaggagat atacatatgg cagatctcaa
     5521 ttggatatcg gccggccacg cgatcgctga cgtcggtacc ctcgagtctg gtaaagaaac
     5581 cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag cttaattaac
     5641 ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     5701 cttgaggggt tttttgctga aacctcaggc atttgagaag cacacggtca cactgcttcc
     5761 ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac gaccctgccc
     5821 tgaaccgacg accgggtcga atttgctttc gaatttctgc cattcatccg cttattatca
     5881 cttattcagg cgtagcaacc aggcgtttaa gggcaccaat aactgcctta aaaaaattac
     5941 gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg
     6001 aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct
     6061 tgcgtataat atttgcccat agtgaaaacg ggggcgaaga agttgtccat attggccacg
     6121 tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca
     6181 ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat
     6241 atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca
     6301 gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg
     6361 tctttcattg ccatacggaa ctccggatga gcattcatca ggcgggcaag aatgtgaata
     6421 aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc
     6481 agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct
     6541 ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta
     6601 gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt
     6661 tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccaaaag
     6721 ttggcccagg gcttcccggt atcaacaggg acaccaggat ttatttattc tgcgaagtga
     6781 tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc caacttactg
     6841 atttagtgta tgatggtgtt tttgaggtgc tccagtggct tctgtttcta tcagctgtcc
     6901 ctcctgttca gctactgacg gggtggtgcg taacggcaaa agcaccgccg gacatcagcg
     6961 ctagcggagt gtatactggc ttactatgtt ggcactgatg agggtgtcag tgaagtgctt
     7021 catgtggcag gagaaaaaag gctgcaccgg tgcgtcagca gaatatgtga tacaggatat
     7081 attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggaaa
     7141 tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt aacagggaag
     7201 tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca agcatcacga
     7261 aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     7321 tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat
     7381 tccgctgtta tggccgcgtt tgtctcattc cacgc
//
