LOCUS       sequence-384621-        5935 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cas4/1, Cas2, leader, repeat and one spacer from A. 
            acidoterrestris (type V-B). Cas4 domain swapped with type U-I Cas4 
            domain from G. sulfurreducens.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5935)
  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 5935)
  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(I-U)/1_2_VB
FEATURES             Location/Qualifiers
     source          1..5935
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     628..647
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(667..744)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     790..809
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        790..808
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    813..836
                     /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             889..911
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(3409..3427)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3423..3470
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3641..4432)
                     /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(4433..4524)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4572..5310)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(join(5519..5935,1..666))
                     /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 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
       61 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
      121 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
      181 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
      241 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
      301 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
      361 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
      421 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
      481 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
      541 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
      601 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
      661 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
      721 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
      781 ttaggaaatt aatacgactc actataggga gagaattgtg agcggataac aattcccctg
      841 tagattaatt aagcggccgc cctgcaggac tcgagttcta gaaataattt tgtttaactt
      901 taagaaggag atatacatat ggctgagaca gacgggagta ttcctctcat cccggttcgc
      961 atgctcaacg aacacgtcta ctgcccgcga ctggcctatc tcatgtgggt gcagggtgag
     1021 ttctcccaca atgagttcac ggttgatggc gttatccgcc accgcagggt cgatgctggc
     1081 ggcggagtgc tgccctccga gacccaggag gattccagga tacatgcccg ctcggtgagt
     1141 ctcagctcgg aacggctggg aattaccgcc aagatcgatc tggtggaagg ggagggagca
     1201 tacgtttctc ctgtcgatta caagcggggc aaacgccccc atgtggccgg cggagcatac
     1261 gagccggagc gggttcagct ctgtgcccag gggcttcttc tgcgggagca cggatttgcc
     1321 agcgatggcg gcgctctcta cttcgttgcc tcccgcgaac gggtgccggt tgcatttgat
     1381 gatgaactga tcggaagaac cctggccgcc attgatgaga tgggacgcac ggcgttgtcg
     1441 ggcacgatgc ccccgccgct ggaggacagt cccaaatgcc ctcgctgctc gctggtgggg
     1501 atctgtctgc cggatgaagt gcgctttctc acgagtccgg acggagagac cgttccgata
     1561 cgagatattg tccccagtcg agatgattta ggaaccgtgt acgtgtcgga gcctgggacc
     1621 cgggtcggaa agcgtggtta cgagttgacc atcacgtcga aagatgggat ggaatcgacc
     1681 gttccgttaa aagatgttcg gcacatctcc ctcttcggga acgttcagat ttccacccaa
     1741 ttggtgcacg attgtatgga gtccggcgtc accatcagct acttgactgc cgccggccgt
     1801 ctcgtcggga tgaaccacaa tttgatttcg aaaaacgtct tggtgcggcg tgaacagttc
     1861 aagcggtttg gggacgagcg tgatcgactg acacttgcga ggtatatcgt tcgcgcgaaa
     1921 attctcaacc agcgcacctt gcttcgtcga aatgcgcacg ggctcgacaa ggtcattttg
     1981 aaagatttgc tcgatcacgc ccgacgttcc gaagaagcca cttccattga ctcgttgctc
     2041 ggcattgaag ggattgcggc gaaacagtac atgcaggcgt ttgtgctgat gctgaaagta
     2101 aaaggcgttc ctgcgggcga aacgctgatg aacggcagaa atcgccgccc accgaaggac
     2161 ccggttaatg cgttgctgtc acttggatat tcgttgttgc aacgggatat gtatgccgcc
     2221 ttggctgcag tgggcctgga ccccttgatg ggcttttacc atcgcattga aacggggcgt
     2281 ccgtcactgg tactcgacat gatggaaccg tttcgagcga tcatcgcgga ttcggtggtc
     2341 gttcgaacat tgaacaccgg cgaaattgcc tgggatgact tttatatcgg gcaggagtcc
     2401 tgttcgttga agctgcctgg acggaaacga ttttttcagg cgtttgagcg acgcatgcac
     2461 gagaccgtga cgcatccggt gttcggttac aaattgagtt accgtcggat gctggaacta
     2521 gaagcgcgct tcttgtcgcg atacttgatg ggcgaattgc cggaatatcg cccaattgtc
     2581 acgaggtgat ggcgatgcgc cgatatgtgc tggtgagcta cgatatcagt gatcaaaaac
     2641 gctggcgaag ggtatacaaa atcgttcgag gctatggcat gcacgttcag tattcggtgt
     2701 tcctatgcca actcaccgat caggatgagg ccgagttgaa gcaattgcta ttggatgtga
     2761 ttcatcagtc gatggatcaa gtcatttttg caaggattgg aaccgtgcag gcgaaagctg
     2821 ccgagcggaa tatgtcgtgc attgggcggg acttcgtacc attggatttg aagggattgg
     2881 tattttgaag gattgatgta aacttcaaat acgcgtaaaa tataatctgt ctagaggaca
     2941 gaatttttca acgggtgtgc caatggccac tttccaggtg gcaaagcccg ttgagcttct
     3001 caaaaagaac gctcgctcag tgttctgacc tttcgagcgc ctgttcaggg cgaaaaccct
     3061 gggaggcgct cgaatcatag gtgggacaag ggattcgcgg cgaaaacgac gggagagaac
     3121 gagaatgaaa cgagatgaaa tcgatgggaa acatcaagtg ctcgtttatg ggcttggatc
     3181 tccgcgtggt tcaagggctg cgagcgcccg ctgtcggatc actgagcgag cgatctgaga
     3241 agtggcactg tttggtaaag gtaaaaagac gaatgatgca tcctaacatt ggaagtggat
     3301 aacggatccg cgatcgcggc gcgccacctg gtggccggcc ggtaccacgc gtgcgcgctg
     3361 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     3421 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaacctc
     3481 aggcatttga gaagcacacg gtcacactgc ttccggtagt caataaaccg gtaaaccagc
     3541 aatagacata agcggctatt taacgaccct gccctgaacc gacgaccggg tcatcgtggc
     3601 cggatcttgc ggcccctcgg cttgaacgaa ttgttagaca ttatttgccg actaccttgg
     3661 tgatctcgcc tttcacgtaa tggacaaatt cttccaactg atctgcgcgc gaggccaagc
     3721 gatcttcttc ttgtccaaga taagcctgtc tagcttcaag tatgacgggc tgatactggg
     3781 ccggcaggcg ctccattgcc cagtcggcag cgacatcctt cggcgcgatt ttgccggtta
     3841 ctgcgctgta ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg ccagcccagt
     3901 cgggcggcga gttccatagc gttaaggttt catttagcgc ctcaaataga tcctgttcag
     3961 gaaccggatc aaagagttcc tccgccgctg gacctaccaa ggcaacgcta tgttctcttg
     4021 cttttgtcag caagatagcc agatcaatgt cgatcgtggc tggctcgaag atacctgcaa
     4081 gaatgtcatt gcgctgccat tctccaaatt gcagttcgcg cttagctgga taacgccacg
     4141 gaatgatgtc gtcgtgcaca acaatggtga cttctacagc gcggagaatc tcgctctctc
     4201 caggggaagc cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa
     4261 gccttacggt caccgtaacc agcaaatcaa tatcactgtg cggcttcagg ccgccatcca
     4321 ctgcggagcc gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc
     4381 caactacctc tgatagttga gtcgatactt cggcgatcac cgcttccctc atactcttcc
     4441 tttttcatta ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4501 aatgtattta gaaaaataaa caaatagcta gctcactcgg tcgctacgct ccgggcgtga
     4561 gactgcggcg ggcgctgcgg acacatacaa agttacccac agattccgtg gataagcagg
     4621 ggactaacat gtgaggcaaa acagcagggc cgcgccggtg gcgtttttcc ataggctccg
     4681 ccctcctgcc agagttcaca taaacagacg cttttccggt gcatctgtgg gagccgtgag
     4741 gctcaaccat gaatctgaca gtacgggcga aacccgacag gacttaaaga tccccaccgt
     4801 ttccggcggg tcgctccctc ttgcgctctc ctgttccgac cctgccgttt accggatacc
     4861 tgttccgcct ttctccctta cgggaagtgt ggcgctttct catagctcac acactggtat
     4921 ctcggctcgg tgtaggtcgt tcgctccaag ctgggctgta agcaagaact ccccgttcag
     4981 cccgactgct gcgccttatc cggtaactgt tcacttgagt ccaacccgga aaagcacggt
     5041 aaaacgccac tggcagcagc cattggtaac tgggagttcg cagaggattt gtttagctaa
     5101 acacgcggtt gctcttgaag tgtgcgccaa agtccggcta cactggaagg acagatttgg
     5161 ttgctgtgct ctgcgaaagc cagttaccac ggttaagcag ttccccaact gacttaacct
     5221 tcgatcaaac cacctcccca ggtggttttt tcgtttacag ggcaaaagat tacgcgcaga
     5281 aaaaaaggat ctcaagaaga tcctttgatc ttttctactg aaccgctctt atttcagtgc
     5341 aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag ttgtaattct
     5401 catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     5461 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     5521 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5581 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     5641 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     5701 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     5761 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
     5821 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     5881 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacat
//
