LOCUS       sequence-234749-        5704 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses concatenated noncoding sequences surrounding the 
            CRISPR-Cas12g1 system in the native genomic loci; expresses the 
            tracrRNA required for activity..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5704)
  AUTHORS   Yan WX, Hunnewell P, Alfonse LE, Carte JM, Keston-Smith E, 
            Sothiselvam S, Garrity AJ, Chong S, Makarova KS, Koonin EV, Cheng 
            DR, Scott DA
  TITLE     Functionally diverse type V CRISPR-Cas systems.
  JOURNAL   Science. 2019 Jan 4;363(6422):88-91. doi: 10.1126/science.aav7271. 
            Epub 2018 Dec 6.
  PUBMED    30523077
REFERENCE   2  (bases 1 to 5704)
  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: 
            "10.1126/science.aav7271"; journalName: "Science"; date: "2019-01-4-
            4"; volume: "363"; issue: "6422"; pages: "88-91"
COMMENT     Sequence Label: pACYC-Cas12g1-noncoding
FEATURES             Location/Qualifiers
     source          1..5704
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(215..232)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(349..894)
                     /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(429..448)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(1006..1011,1012..1028,1029..1034)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             1082..2272
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(1434..1453)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2711..2730
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2711..2729
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2730..2754
                     /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)."
     promoter        join(2763..2768,2769..2786,2787..2792)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4083..4101)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4097..4144
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4765..5424)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5339..5358
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5425..5527)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 ggcttctgtt tctatcagct gtccctcctg ttcagctact gacggggtgg tgcgtaacgg
       61 caaaagcacc gccggacatc agcgctagcg gagtgtatac tggcttacta tgttggcact
      121 gatgagggtg tcagtgaagt gcttcatgtg gcaggagaaa aaaggctgca ccggtgcgtc
      181 agcagaatat gtgatacagg atatattccg cttcctcgct cactgactcg ctacgctcgg
      241 tcgttcgact gcggcgagcg gaaatggctt acgaacgggg cggagatttc ctggaagatg
      301 ccaggaagat acttaacagg gaagtgagag ggccgcggca aagccgtttt tccataggct
      361 ccgcccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac
      421 aggactataa agataccagg cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc
      481 tgcctttcgg tttaccggtg tcattccgct gttatggccg cgtttgtctc attccacgcc
      541 tgacactcag ttccgggtag gcagttcgct ccaagctgga ctgtatgcac gaaccccccg
      601 ttcagtccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggaaagac
      661 atgcaaaagc accactggca gcagccactg gtaattgatt tagaggagtt agtcttgaag
      721 tcatgcgccg gttaaggcta aactgaaagg acaagttttg gtgactgcgc tcctccaagc
      781 cagttacctc ggttcaaaga gttggtagct cagagaacct tcgaaaaacc gccctgcaag
      841 gcggtttttt cgttttcaga gcaagagatt acgcgcagac caaaacgatc tcaagaagat
      901 catcttatta atcagataaa atatttctag atttcagtgc aatttatctc ttcaaatgta
      961 gcacctgaag tcagccccat acgatataag ttgtaattct catgtttgac agcttatcat
     1021 cgataagctt taatgcggta gtttatcaca gttaaattgc taacgcagtc aggcaccgtg
     1081 tatgaaatct aacaatgcgc tcatcgtcat cctcggcacc gtcaccctgg atgctgtagg
     1141 cataggcttg gttatgccgg tactgccggg cctcttgcgg gatatcgtcc attccgacag
     1201 catcgccagt cactatggcg tgctgctagc gctatatgcg ttgatgcaat ttctatgcgc
     1261 acccgttctc ggagcactgt ccgaccgctt tggccgccgc ccagtcctgc tcgcttcgct
     1321 acttggagcc actatcgact acgcgatcat ggcgaccaca cccgtcctgt ggatcctcta
     1381 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat
     1441 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt
     1501 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca
     1561 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct
     1621 aatgcaggag tcgcataagg gagagcgtcg accgatgccc ttgagagcct tcaacccagt
     1681 cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt
     1741 tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg
     1801 ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc
     1861 cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat
     1921 tatcgccggc atggcggccg acgcgctggg ctacgtcttg ctggcgttcg cgacgcgagg
     1981 ctggatggcc ttccccatta tgattcttct cgcttccggc ggcatcggga tgcccgcgtt
     2041 gcaggccatg ctgtccaggc aggtagatga cgaccatcag ggacagcttc aaggatcgct
     2101 cgcggctctt accagcctaa cttcgatcat tggaccgctg atcgtcacgg cgatttatgc
     2161 cgcctcggcg agcacatgga acgggttggc atggattgta ggcgccgccc tataccttgt
     2221 ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct gaatggaagc
     2281 cggcggcacc tcgctaacgg attcaccact ccaagaattg gagccaatca attcttgcgg
     2341 agaactgtga atgcgcaaac caacccttgg cagaacatat ccatcgcgtc cgccatctcc
     2401 agcagccgca cgcggcgcat ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc
     2461 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
     2521 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
     2581 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtcc
     2641 cctacgtgct gctgaagttg cccgcaacag agagtggaac caaccggtga taccacgata
     2701 ctatgactga taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     2761 gatttacact ttatgcttcc ggctcgtatg tttctagact cgagtgcatc ccggacgatt
     2821 tcaaacgcct ggtgctgccc gtgttcgctc accgcatcgt ggtgaatacc cgctacgttt
     2881 ccacacagaa aaaatccgaa caggcggaag ccatcctgcg cgaaattctc gactccaccc
     2941 ccgtgcccct gtaggccctg aagtcctttg tggtatattg ctcacaacag taacagtcat
     3001 ggcccaggca tcgtccacac ctgcagtttc tccccggcca cggccgcgct accgcgagga
     3061 gcgcaccctg gtccgcaagc tgctgccccg cccgggccag tcgaagcagg agttccgcga
     3121 aaacgtaaag aagctgcgca aggcatttaa agacaacaaa caatgggaaa agaataaaaa
     3181 aagaggtctt ttcaaatgtg aggggtgtgg gtcccaagcc caggtagatg aaaatgcggc
     3241 tcgcgtgctc ggccgggtat tctggggcga aatcgagctg ccgaccgcaa tcccctgacg
     3301 acccttgacg gaactggccg atttgctaaa atatttttga tgcttactta gtcatctggt
     3361 tggcaaacct ccgcggacct tcgggaccaa tggagaggaa cccagccgag aagcatcgag
     3421 ccggtaaatg ccggaaattt ttgagtcagg agaagctaac ttcttcaaac tcaggccctt
     3481 ggagccggtc agaaacacgc gagcaaaagc cggcctctca gctacgcaca ccacgacgaa
     3541 ccaaacctgc cggggctact ccaggccgtc ctttcgctgg cggacgcagc cgcactgacc
     3601 acgggcacac tcccgcgaac aggagaaatt cgtcccggca gaaaatgcag ggggatcggc
     3661 tccccagagg ctcgcgtgca gcgcagtgaa gaagttcatc cagacgcaaa aagcggcgag
     3721 aagcgcaagc cgggcgtgtg ggtccagccg gcccgcgcat gaagactccg ctgtgcgagt
     3781 ccagtgttcg gctggtgtgc aggaaacaga gtcagcacag atacgccaac agacagcaac
     3841 tgtgtgattt tcgtctcggg gccgggctga gataatcctg gccgatgagg tctgctgtcc
     3901 agcagtggtg ggcaagcgcg gatcgagcgg gctggcgcag ttttgcgggc gcgctggcgg
     3961 ctctgggagt ggcgctgctg ctggcgctgt attcttccgc agcggcggag ggcgggcaga
     4021 agtggcgggc atggctcgag ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     4081 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     4141 tttgctgaaa ggaggaacta tatccggatg agtcaacgcc atgagcggcc tcatttctta
     4201 ttctgagtta caacagtccg caccgctgcc ggtagctcct tccggtgggc gcggggcatg
     4261 actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg
     4321 gcagcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     4381 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
     4441 cctacatctg tattaacgaa gcgctaaccg tttttatcag gctctgggag gcagaataaa
     4501 tgatcatatc gtcaattatt acctccacgg ggagagcctg agcaaactgg cctcaggcat
     4561 ttgagaagca cacggtcaca ctgcttccgg tagtcaataa accggtaaac cagcaataga
     4621 cataagcggc tatttaacga ccctgccctg aaccgacgac cgggtcgaat ttgctttcga
     4681 atttctgcca ttcatccgct tattatcact tattcaggcg tagcaaccag gcgtttaagg
     4741 gcaccaataa ctgccttaaa aaaattacgc cccgccctgc cactcatcgc agtactgttg
     4801 taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat
     4861 cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg
     4921 ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg
     4981 attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc
     5041 accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta
     5101 ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg
     5161 aacactatcc catatcacca gctcaccgtc tttcattgcc atacggaatt ccggatgagc
     5221 attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt
     5281 tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc
     5341 aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt
     5401 atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc
     5461 aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg
     5521 ccgatcaacg tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac
     5581 accaggattt atttattctg cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag
     5641 tgcgtcgggt gatgctgcca acttactgat ttagtgtatg atggtgtttt tgaggtgctc
     5701 cagt
//
