LOCUS       sequence-384673-        6008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cas4/1, 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 6008)
  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 6008)
  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-4/1_2_VB
FEATURES             Location/Qualifiers
     source          1..6008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     527..546
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(566..643)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     689..708
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        689..707
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    712..735
                     /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             788..810
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(3314..3332)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3328..3375
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3546..4337)
                     /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(4338..4429)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(join(5491..6008,1..565))
                     /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 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
       61 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
      121 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
      181 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
      241 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
      301 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
      361 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
      421 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
      481 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
      541 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
      601 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
      661 cccttatgcg actcctgcat taggaaatta atacgactca ctatagggag agaattgtga
      721 gcggataaca attcccctgt agattaatta agcggccgcc ctgcaggact cgagttctag
      781 aaataatttt gtttaacttt aagaaggaga tatacatatg gccgtaccga ttcggatgct
      841 gaatgaactc tcttattgcg aacgtttata tcatttgatg cacgtgcaag ggttattcga
      901 aaaatctgcg gacacagtgg aaggcagcgg tcagcacgac cgggcggaac gccgcaaaag
      961 accatcagac gtggggcatg atgtgtggga tattgcaccg cagagcctcc atctcggcga
     1021 cgaggatttg gggattgtgg gcaaattgga tgcgattcgc tatcaggacg acgggtattg
     1081 ggagcccgtt gaagcgaaac attcaagcgc accaaacggt ggcacttcat ttcgcctcgg
     1141 cgagtgggaa ttggacggca aggcgtggcc gaatgaccag atacagttgt gtgcacaagg
     1201 attgttactg agagccaacg gatttccgac cattgcgggt aagttgttct accgaggaaa
     1261 cagacagacg gtaaggattg tattttcgga tgatttgatt gctgcaacgg ttgccaccat
     1321 ccggagagca cacgagttgg aacaggcttc gatgccctcg ccactgctga attccgacaa
     1381 gtgcttccga tgctcgttga atggtgtctg tcttcctgac gaaacgattc gcttgacgag
     1441 tccggacgga gagaccgttc cgatacgaga tattgtcccc agtcgagatg atttaggaac
     1501 cgtgtacgtg tcggagcctg ggacccgggt cggaaagcgt ggttacgagt tgaccatcac
     1561 gtcgaaagat gggatggaat cgaccgttcc gttaaaagat gttcggcaca tctccctctt
     1621 cgggaacgtt cagatttcca cccaattggt gcacgattgt atggagtccg gcgtcaccat
     1681 cagctacttg actgccgccg gccgtctcgt cgggatgaac cacaatttga tttcgaaaaa
     1741 cgtcttggtg cggcgtgaac agttcaagcg gtttggggac gagcgtgatc gactgacact
     1801 tgcgaggtat atcgttcgcg cgaaaattct caaccagcgc accttgcttc gtcgaaatgc
     1861 gcacgggctc gacaaggtca ttttgaaaga tttgctcgat cacgcccgac gttccgaaga
     1921 agccacttcc attgactcgt tgctcggcat tgaagggatt gcggcgaaac agtacatgca
     1981 ggcgtttgtg ctgatgctga aagtaaaagg cgttcctgcg ggcgaaacgc tgatgaacgg
     2041 cagaaatcgc cgcccaccga aggacccggt taatgcgttg ctgtcacttg gatattcgtt
     2101 gttgcaacgg gatatgtatg ccgccttggc tgcagtgggc ctggacccct tgatgggctt
     2161 ttaccatcgc attgaaacgg ggcgtccgtc actggtactc gacatgatgg aaccgtttcg
     2221 agcgatcatc gcggattcgg tggtcgttcg aacattgaac accggcgaaa ttgcctggga
     2281 tgacttttat atcgggcagg agtcctgttc gttgaagctg cctggacgga aacgattttt
     2341 tcaggcgttt gagcgacgca tgcacgagac cgtgacgcat ccggtgttcg gttacaaatt
     2401 gagttaccgt cggatgctgg aactagaagc gcgcttcttg tcgcgatact tgatgggcga
     2461 attgccggaa tatcgcccaa ttgtcacgag gtgatggcga tgcgccgata tgtgctggtg
     2521 agctacgata tcagtgatca aaaacgctgg cgaagggtat acaaaatcgt tcgaggctat
     2581 ggcatgcacg ttcagtattc ggtgttccta tgccaactca ccgatcagga tgaggccgag
     2641 ttgaagcaat tgctattgga tgtgattcat cagtcgatgg atcaagtcat ttttgcaagg
     2701 attggaaccg tgcaggcgaa agctgccgag cggaatatgt cgtgcattgg gcgggacttc
     2761 gtaccattgg atttgaaggg attggtattt tgaaggattg atgtaaactt caaatacgcg
     2821 taaaatataa tctgtctaga ggacagaatt tttcaacggg tgtgccaatg gccactttcc
     2881 aggtggcaaa gcccgttgag cttctcaaaa agaacgctcg ctcagtgttc tgacctttcg
     2941 agcgcctgtt cagggcgaaa accctgggag gcgctcgaat cataggtggg acaagggatt
     3001 cgcggcgaaa acgacgggag agaacgagaa tgaaacgaga tgaaatcgat gggaaacatc
     3061 aagtgctcgt ttatgggctt ggatctccgc gtggttcaag ggctgcgagc gcccgctgtc
     3121 ggatcactga gcgagcgatc tgagaagtgg cactgtttgg taaaggtaaa aagacgaatg
     3181 atgcatccta acattggaag tggataacgg atccgcgatc gcggcgcgcc acctggtggc
     3241 cggccggtac cacgcgtgcg cgctgatccg gctgctaaca aagcccgaaa ggaagctgag
     3301 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     3361 ttgaggggtt ttttgctgaa acctcaggca tttgagaagc acacggtcac actgcttccg
     3421 gtagtcaata aaccggtaaa ccagcaatag acataagcgg ctatttaacg accctgccct
     3481 gaaccgacga ccgggtcatc gtggccggat cttgcggccc ctcggcttga acgaattgtt
     3541 agacattatt tgccgactac cttggtgatc tcgcctttca cgtaatggac aaattcttcc
     3601 aactgatctg cgcgcgaggc caagcgatct tcttcttgtc caagataagc ctgtctagct
     3661 tcaagtatga cgggctgata ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca
     3721 tccttcggcg cgattttgcc ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact
     3781 acatttcgct catcgccagc ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt
     3841 agcgcctcaa atagatcctg ttcaggaacc ggatcaaaga gttcctccgc cgctggacct
     3901 accaaggcaa cgctatgttc tcttgctttt gtcagcaaga tagccagatc aatgtcgatc
     3961 gtggctggct cgaagatacc tgcaagaatg tcattgcgct gccattctcc aaattgcagt
     4021 tcgcgcttag ctggataacg ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct
     4081 acagcgcgga gaatctcgct ctctccaggg gaagccgaag tttccaaaag gtcgttgatc
     4141 aaagctcgcc gcgttgtttc atcaagcctt acggtcaccg taaccagcaa atcaatatca
     4201 ctgtgyggct tcaggccgcc atccactgcg gagccgtaca aatgtacggc cagcaacgtc
     4261 ggttcgagat ggcgctcgat gacgccaact acctctgata gttgagtcga tacttcggcg
     4321 atcaccgctt ccctcatact cttccttttt cattattatt gaagcattta tcagggttat
     4381 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat agctagctca
     4441 ctcggtcgct acgctccggg cgtgagactg cggcgggcgc tgcggacaca tacaaagtta
     4501 cccacagatt ccgtggataa gcaggggact aacatgtgag gcaaaacagc agggccgcgc
     4561 cggtggcgtt tttccatagg ctccgccctc ctgccagagt tcacataaac agacgctttt
     4621 ccggtgcatc tgtgggagcc gtgaggctca accatgaatc tgacagtacg ggcgaaaccc
     4681 gacaggactn taaagatmcc mrscgtttcc ssckggwmgc tccctcktgc gctctcctgt
     4741 tccgaccctg ccgyttaccg gatacctgtn ccgcctttct cccttncggg aagygtggcg
     4801 ctttctcata gctcacrcws tnggtatctc rgytcggtgt aggtcgttcg ctccaagctg
     4861 ggctgtrwgc amgaacyccc cgttcagccc gacygctgcg ccttatccgg taactrtysw
     4921 cttgagtcca acccggwaar rcacgrywwa wcgccactgg cagcagccay tggtaacwgg
     4981 rwkttcgcag aggaktwgkt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt
     5041 ccggctacac tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacggt
     5101 taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg
     5161 aaggttaagt cagttgggga actgcttaac cttcgatcaa accacctccc caggtggttt
     5221 tttcgtttac agggcaaaag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5281 tcttttctac tgaaccgctc ttatttcagt gcaatttatc tcttcaaatg tagcacctga
     5341 agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg
     5401 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     5461 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5521 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5581 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     5641 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     5701 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     5761 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     5821 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     5881 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     5941 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     6001 agaactta
//
