LOCUS       sequence-384674-        5657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cas4 (K70A), Cas1, Cas2, leader, repeat and one spacer from 
            F. Novicida (type V-A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5657)
  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 5657)
  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_VA
FEATURES             Location/Qualifiers
     source          1..5657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        800..891
                     /gene="bla"
                     /label=AmpR promoter
     CDS             892..1683
                     /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"
     terminator      complement(1854..1901)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1897..1915
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     RBS             4070..4092
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4145..4168
                     /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)."
     primer_bind     complement(4172..4191)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4173..4191)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        4237..4314
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4315..5397
                     /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     complement(4334..4353)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtaaacgaaa aaaccacctg gggaggtggt ttgatcgaag gttaagcagt tccccaactg
       61 acttaacctt cgaaaaaacc acctggggag gtggtttgat cgaaggttaa gtcagttggg
      121 gaactgctta accgtggtaa ctggctttcg cagagcacag caaccaaatc tgtccttcca
      181 gtgtagccgg actttggcgc acacttcaag agcaaccgcg tgtttagcta aacaaatcct
      241 ctgcgaactc ccagttacca atggctgctg ccagtggcgt tttaccgtgc ttttccgggt
      301 tggactcaag tgaacagtta ccggataagg cgcagcagtc gggctgaacg gggagttctt
      361 gcttacagcc cagcttggag cgaacgacct acaccgagcc gagataccag tgtgtgagct
      421 atgagaaagc gccacacttc ccgtaaggga gaaaggcgga acaggtatcc ggtaaacggc
      481 agggtcggaa caggagagcg caagagggag cgacccgccg gaaacggtgg ggatctttaa
      541 gtcctgtcgg gtttcgcccg tactgtcaga ttcatggttg agcctcacgg ctcccacaga
      601 tgcaccggaa aagcgtctgt ttatgtgaac tctggcagga gggcggagcc tatggaaaaa
      661 cgccaccggc gcggccctgc tgttttgcct cacatgttag tcccctgctt atccacggaa
      721 tctgtgggta actttgtatg tgtccgcagc gcccgccgca gtctcacgcc cggagcgtag
      781 cgaccgagtg agctagctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
      841 atgagacaat aaccctgata aatgcttcaa taataatgaa aaaggaagag tatgagggaa
      901 gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat
      961 ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag
     1021 ccgcacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg
     1081 cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc
     1141 cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct
     1201 aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg tatcttcgag
     1261 ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt
     1321 gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt
     1381 gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag
     1441 cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg
     1501 ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc
     1561 atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca
     1621 gatcagttgg aagaatttgt ccattacgtg aaaggcgaga tcaccaaggt agtcggcaaa
     1681 taatgtctaa caattcgttc aagccgaggg gccgcaagat ccggccacga tgacccggtc
     1741 gtcggttcag ggcagggtcg ttaaatagcc gcttatgtct attgctggtt taccggttta
     1801 ttgactaccg gaagcagtgt gaccgtgtgc ttctcaaatg cctgaggttt cagcaaaaaa
     1861 cccctcaaga cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca
     1921 gcagccaact cagcttcctt tcgggctttg ttagcagccg gatcagcgcg cacgcgtggt
     1981 accggccggc caccaggtgg cgcgccgcga tcgcggatcc gttatccact tccaatgtta
     2041 ttttaacagt ggccttatta aatgacttct catctacaac agtagaaatt atttaaagtt
     2101 cttagacccg tttttgccta aatcagcaaa aaaagaccta aaatctatga tttttttagc
     2161 attttgatat ataacaacag tcatattttt aaatatctct aagaacttag tagttatttt
     2221 aacataaaga ctaaggtttg caatttttag tctataaaaa caacctcttg ttctctatga
     2281 atcgcataac catattttat cacatcttta tctggagcat taaagattaa aacactatca
     2341 gcatttgtaa atagtggtac atagttattt tctatctcag ctaaaatcaa gtctaacatt
     2401 ctcttgctat atttgagctc aaataccgaa tattgtaaac gtacaccata actttctaga
     2461 aatttggcaa actttgcacg tactttatta ttactaaaat cataactgac tatcaacata
     2521 caccctccta gtttcataac aaaatattgg atactctgca atttctttgc ctcgcataaa
     2581 ggcacgataa aattctctta catattcaaa tatctctagc ttgtagctga taatctcggc
     2641 aaaaagtatt tttgagtagt cttttgtttt atcttttttt atataaaact gctcattttt
     2701 gcaaccaaaa tcctctacag aaaattgccc gagattccat gattttctaa cttggttatc
     2761 cactatacat ctaaatggct caacaaagtc acaaactagg gatttacgct tataccaagt
     2821 ctgatgacaa aagcccttgt ataaatcaaa gccaaaaagt cgcaaaaaac actctacaaa
     2881 attaaacaac atactatagc ccaagtctaa aacaacatta tatggatcct gttttattct
     2941 aggtttgcgc ccttgccaac tatctaaatg tccatagaat cctttgaaga aatttttggc
     3001 aacattgccc tctataccca ttagctccgc taggcttgtt gtattttcta actctgctat
     3061 tagctgttta ttctttttga ctatctcttg ctgctctgga gttttatctc ttaggctttt
     3121 gactaagtta tgttgattgc gtatctttga tgttataaaa atccttgcta aacttatatc
     3181 ttgctcaact acactatatt gcttatatct agctaaaaaa ttagcctcag caatatcacc
     3241 aaaacataga tatggtctaa agctattttt cataaaaact agctgtatag catttttctt
     3301 acaggtctct aaaagttgcg aggtcatcgt catattacct acgataaaca atgcaagaac
     3361 tttatgaaca gaaagcttag ttttcacctc atcagtttct ttatctttta taactatatt
     3421 ccccaatgat agactaagtt tcactccatc aaaaatatta acaaaaacta tattctttga
     3481 ttcaatatca tttttactaa acataatcta caagtccgtt ttatcacata agtttgcata
     3541 tatacaaaat ttacactttt caatattttg cctaaatgag ttatccattg gattatattg
     3601 cttgattgtt tgaatatgtt tttcaaactt ttctaactca gctgaagttg gtatagctat
     3661 tgggtatgat ttattatcaa ccatcgaata aaatttaatg gctttgacat catagcccat
     3721 ctcttggaga caaaaatatt gcgcataaag ctgatattta tagccatcat agatagtctt
     3781 gatttgcctc gctctctcca caagtaagcc cttatcttta tgaaaaacat caattttacc
     3841 aaccaaacca tatttatggt taataacatc gataccgatc aggtcatctt ttttggtact
     3901 atagattcct ttatcgatac ttttatgagc atcgcgccct tttgtttggc tagatgaata
     3961 gtatgtagta tctgaggcat tactatagat attatgcaaa tatatcgagt aaggacagaa
     4021 tataaaatca tttaaccatg ttatcaaaat ataattttcc atatgtatat ctccttctta
     4081 aagttaaaca aaattatttc tagaactcga gtcctgcagg gcggccgctt aattaatcta
     4141 caggggaatt gttatccgct cacaattctc tccctatagt gagtcgtatt aatttcctaa
     4201 tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc
     4261 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     4321 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     4381 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     4441 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     4501 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     4561 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     4621 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     4681 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     4741 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     4801 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     4861 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     4921 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     4981 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     5041 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     5101 cgcgttggtg cggacatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     5161 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     5221 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     5281 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     5341 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     5401 caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt
     5461 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta acatgagaat
     5521 tacaacttat atcgtatggg gctgacttca ggtgctacat ttgaagagat aaattgcact
     5581 gaaataagag cggttcagta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5641 gcgtaatctt ttgccct
//
