LOCUS       sequence-384627-        6006 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cas4(K81A)/1, Cas2, leader, repeat and one spacer from A. 
            acidoterrestris (type V-B).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6006)
  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 6006)
  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: pCas-mut4/1_2_VB
FEATURES             Location/Qualifiers
     source          1..6006
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     359..378
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(398..475)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     521..540
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        521..539
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    544..567
                     /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)."
     RBS             620..642
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(3146..3164)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3160..3207
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3378..4169)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(4170..4261)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(join(5321..6006,1..397))
                     /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 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
       61 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
      121 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
      181 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
      241 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
      301 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
      361 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
      421 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
      481 tctcgacgct ctcccttatg cgactcctgc attaggaaat taatacgact cactataggg
      541 agagaattgt gagcggataa caattcccct gtagattaat taagcggccg ccctgcagga
      601 ctcgagttct agaaataatt ttgtttaact ttaagaagga gatatacata tggccgtacc
      661 gattcggatg ctgaatgaac tctcttattg cgaacgttta tatcatttga tgcacgtgca
      721 agggttattc gaaaaatctg cggacacagt ggaaggcagc ggtcagcacg accgggcgga
      781 acgccgcaaa agaccatcag acgtggggca tgatgtgtgg gatattgcac cgcagagcct
      841 ccatctcggc gacgaggatt tggggattgt gggcaaattg gatgcgattc gctatcagga
      901 cgacgggtat tgggagcccg ttgaagcggc acattcaagc gcaccaaacg gtggcacttc
      961 atttcgcctc ggcgagtggg aattggacgg caaggcgtgg ccgaatgacc agatacagtt
     1021 gtgtgcacaa ggattgttac tgagagccaa cggatttccg accattgcgg gtaagttgtt
     1081 ctaccgagga aacagacaga cggtaaggat tgtattttcg gatgatttga ttgctgcaac
     1141 ggttgccacc atccggagag cacacgagtt ggaacaggct tcgatgccct cgccactgct
     1201 gaattccgac aagtgcttcc gatgctcgtt gaatggtgtc tgtcttcctg acgaaacgat
     1261 tcgcttgacg agtccggacg gagagaccgt tccgatacga gatattgtcc ccagtcgaga
     1321 tgatttagga accgtgtacg tgtcggagcc tgggacccgg gtcggaaagc gtggttacga
     1381 gttgaccatc acgtcgaaag atgggatgga atcgaccgtt ccgttaaaag atgttcggca
     1441 catctccctc ttcgggaacg ttcagatttc cacccaattg gtgcacgatt gtatggagtc
     1501 cggcgtcacc atcagctact tgactgccgc cggccgtctc gtcgggatga accacaattt
     1561 gatttcgaaa aacgtcttgg tgcggcgtga acagttcaag cggtttgggg acgagcgtga
     1621 tcgactgaca cttgcgaggt atatcgttcg cgcgaaaatt ctcaaccagc gcaccttgct
     1681 tcgtcgaaat gcgcacgggc tcgacaaggt cattttgaaa gatttgctcg atcacgcccg
     1741 acgttccgaa gaagccactt ccattgactc gttgctcggc attgaaggga ttgcggcgaa
     1801 acagtacatg caggcgtttg tgctgatgct gaaagtaaaa ggcgttcctg cgggcgaaac
     1861 gctgatgaac ggcagaaatc gccgcccacc gaaggacccg gttaatgcgt tgctgtcact
     1921 tggatattcg ttgttgcaac gggatatgta tgccgccttg gctgcagtgg gcctggaccc
     1981 cttgatgggc ttttaccatc gcattgaaac ggggcgtccg tcactggtac tcgacatgat
     2041 ggaaccgttt cgagcgatca tcgcggattc ggtggtcgtt cgaacattga acaccggcga
     2101 aattgcctgg gatgactttt atatcgggca ggagtcctgt tcgttgaagc tgcctggacg
     2161 gaaacgattt tttcaggcgt ttgagcgacg catgcacgag accgtgacgc atccggtgtt
     2221 cggttacaaa ttgagttacc gtcggatgct ggaactagaa gcgcgcttct tgtcgcgata
     2281 cttgatgggc gaattgccgg aatatcgccc aattgtcacg aggtgatggc gatgcgccga
     2341 tatgtgctgg tgagctacga tatcagtgat caaaaacgct ggcgaagggt atacaaaatc
     2401 gttcgaggct atggcatgca cgttcagtat tcggtgttcc tatgccaact caccgatcag
     2461 gatgaggccg agttgaagca attgctattg gatgtgattc atcagtcgat ggatcaagtc
     2521 atttttgcaa ggattggaac cgtgcaggcg aaagctgccg agcggaatat gtcgtgcatt
     2581 gggcgggact tcgtaccatt ggatttgaag ggattggtat tttgaaggat tgatgtaaac
     2641 ttcaaatacg cgtaaaatat aatctgtcta gaggacagaa tttttcaacg ggtgtgccaa
     2701 tggccacttt ccaggtggca aagcccgttg agcttctcaa aaagaacgct cgctcagtgt
     2761 tctgaccttt cgagcgcctg ttcagggcga aaaccctggg aggcgctcga atcataggtg
     2821 ggacaaggga ttcgcggcga aaacgacggg agagaacgag aatgaaacga gatgaaatcg
     2881 atgggaaaca tcaagtgctc gtttatgggc ttggatctcc gcgtggttca agggctgcga
     2941 gcgcccgctg tcggatcact gagcgagcga tctgagaagt ggcactgttt ggtaaaggta
     3001 aaaagacgaa tgatgcatcc taacattgga agtggataac ggatccgcga tcgcggcgcg
     3061 ccacctggtg gccggccggt accacgcgtg cgcgctgatc cggctgctaa caaagcccga
     3121 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     3181 tctaaacggg tcttgagggg ttttttgctg aaacctcagg catttgagaa gcacacggtc
     3241 acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc ggctatttaa
     3301 cgaccctgcc ctgaaccgac gaccgggtca tcgtggccgg atcttgcggc ccctcggctt
     3361 gaacgaattg ttagacatta tttgccgact accttggtga tctcgccttt cacgtaatgg
     3421 acaaattctt ccaactgatc tgcgcgcgag gccaagcgat cttcttcttg tccaagataa
     3481 gcctgtctag cttcaagtat gacgggctga tactgggccg gcaggcgctc cattgcccag
     3541 tcggcagcga catccttcgg cgcgattttg ccggttactg cgctgtacca aatgcgggac
     3601 aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt
     3661 aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc
     3721 gccgctggac ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa gatagccaga
     3781 tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg ctgccattct
     3841 ccaaattgca gttcgcgctt agctggataa cgccacggaa tgatgtcgtc gtgcacaaca
     3901 atggtgactt ctacagcgcg gagaatctcg ctctctccag gggaagccga agtttccaaa
     3961 aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc
     4021 aaatcaatat cactgtgcgg cttcaggccg ccatccactg cggagccgta caaatgtacg
     4081 gccagcaacg tcggttcgag atggcgctcg atgacgccaa ctacctctga tagttgagtc
     4141 gatacttcgg cgatcaccgc ttccctcata ctcttccttt ttcattatta ttgaagcatt
     4201 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4261 atagctagct cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca
     4321 catacaaagt tacccacaga ttccgtggat aagcagggga ctaacatgtg aggcaaaaca
     4381 gcagggccgc gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa
     4441 acagacgctt ttccggtgca tctgtgggag ccgtgaggct caaccatgaa tctgacagta
     4501 cgggcgaaac ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg
     4561 cgctctcctg ttccgaccct gccgtttacc ggatacctgt tccgcctttc tcccttacgg
     4621 gaagtgtggc gctttctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttcg
     4681 ctccaagctg ggctgtaagc aagaactccc cgttcagccc gactgctgcg ccttatccgg
     4741 taactgttca cttgagtcca acccggaaaa gcacggtaaa acgccactgg cagcagccat
     4801 tggtaactgg gagttcgcag aggatttgtt tagctaaaca cgcggttgct cttgaagtgt
     4861 gcgccaaagt ccggctacac tggaaggaca gatttggttg ctgtgctctg cgaaagccag
     4921 ttaccacggt taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt
     4981 ggttttttcg aaggttaagt cagttgggga actgcttaac cttcgatcaa accacctccc
     5041 caggtggttt tttcgtttac agggcaaaag attacgcgca gaaaaaaagg atctcaagaa
     5101 gatcctttga tcttttctac tgaaccgctc ttatttcagt gcaatttatc tcttcaaatg
     5161 tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg
     5221 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     5281 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5341 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5401 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     5461 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     5521 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     5581 cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc
     5641 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     5701 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     5761 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     5821 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     5881 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     5941 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     6001 caatgg
//
