LOCUS       sequence-255045-        4979 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of T. thermophilus Csm complex in E. coli (crRNA).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4979)
  AUTHORS   Liu TY, Liu JJ, Aditham AJ, Nogales E, Doudna JA
  TITLE     Target preference of Type III-A CRISPR-Cas complexes at the 
            transcription bubble.
  JOURNAL   Nat Commun. 2019 Jul 5;10(1):3001. doi: 10.1038/s41467-019-10780-2.
  PUBMED    31278272
REFERENCE   2  (bases 1 to 4979)
  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"; journalName: "Nat 
            Commun."; date: "2019-07-5"; volume: "10(1):3001. doi"; pages: " 
            10.1038/s41467-019-10780-2"
COMMENT     Sequence Label: pACYC-TtCas6-4xcrRNA4.5
FEATURES             Location/Qualifiers
     source          1..4979
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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        517..535
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    536..560
                     /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)."
     CDS             1335..1379
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1417..1435)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1431..1478
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1699..2358)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2273..2292
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2359..2461)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2853..2870)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2987..3532)
                     /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(3067..3086)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3757..4839)
                     /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     4801..4820
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4840..4917)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4963..4979,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggttgca agggattgag ccccgtaagg ggattgcgac ccgtagataa
      121 ggcgcccggg gacgaccacg tcaaggcgca gttgcaaggg attgagcccc gtaaggggat
      181 tgcgacccgt agataaggcg cccggggacg accacgtcaa ggcgcagttg caagggattg
      241 agccccgtaa ggggattgcg acccgtagat aaggcgcccg gggacgacca cgtcaaggcg
      301 cagttgcaag ggattgagcc ccgtaagggg attgcgaccc gtagataagg cgcccgggga
      361 cgaccacgtc aaggcgcagt tgcaagggat tgagccccgt aaggggattg cgacgaattc
      421 gagctcggcg cgcctgcagg tcgacaagct tgcggccgca taatgcttaa gtcgaacaga
      481 aagtaatcgt attgtacacg gccgcataat cgaaattaat acgactcact ataggggaat
      541 tgtgagcgga taacaattcc ccatcttagt atattagtta agtataagaa ggagatatac
      601 atatggttct ggcagcactg gttctggtgc tggaaggtga aggtctgccg gaaccgctgg
      661 gtctgcgtgg ttttttctat ggtctgctgc gtgaagttgc accggaagtt catgatcagg
      721 gtgaaaatcc gtttgcactg ggttttggtg gtcgtgaagg tgcagcatgg gcacgtgtta
      781 gcctgctggt tgaaggcctg tatgcacgtc tggcaccgcg tctgtatgcc ctggaaggcg
      841 aagaagttcg tctgggtccg ccttttcgtg ttcgtgcagt tctgcaagaa ggtcatcctt
      901 gggcaggcgt tagcacctat cctcgtctgt ttcagggtcc tccgagccgt gatctggcac
      961 tgcgttttgc aagcccgacc ttttttcgtc gtaaaggtgt tcattatccg gttccagaac
     1021 ctcgtctggt actggaaagc ctgctgcgtc gtctggaagc atttggtccg ctgaaagctc
     1081 cggaaggtgt tcgtgaagca ctgctggaac gtaccaccgt tcgtagcctg gaaggtcgta
     1141 ccctgcctgc acgtaccgaa gttgataccg caggttttgt gggtcgtgtt gtttatcatc
     1201 tgcctcgtgc aaccgaagaa gaggcacttt ggctgagcgc actgggtcgt tttgcatttt
     1261 atagcggtgt tggtgcaaaa accagcctgg gttatggtcg tgcacgtgcc gaaagcgcat
     1321 aactcgagtc tggtaaagaa accgctgctg cgaaatttga acgccagcac atggactcgt
     1381 ctactagcgc agcttaatta acctaggctg ctgccaccgc tgagcaataa ctagcataac
     1441 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaacctcag gcatttgaga
     1501 agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa tagacataag
     1561 cggctattta acgaccctgc cctgaaccga cgaccgggtc gaatttgctt tcgaatttct
     1621 gccattcatc cgcttattat cacttattca ggcgtagcaa ccaggcgttt aagggcacca
     1681 ataactgcct taaaaaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc
     1741 attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag
     1801 cggcatcagc accttgtcgc cttgcgtata atatttgccc atagtgaaaa cgggggcgaa
     1861 gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc
     1921 tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta
     1981 acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact
     2041 ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact
     2101 atcccatatc accagctcac cgtctttcat tgccatacgg aactccggat gagcattcat
     2161 caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt
     2221 ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga
     2281 ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc
     2341 agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa
     2401 tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc
     2461 aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg
     2521 atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc
     2581 gggtgatgct gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg
     2641 cttctgtttc tatcagctgt ccctcctgtt cagctactga cggggtggtg cgtaacggca
     2701 aaagcaccgc cggacatcag cgctagcgga gtgtatactg gcttactatg ttggcactga
     2761 tgagggtgtc agtgaagtgc ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag
     2821 cagaatatgt gatacaggat atattccgct tcctcgctca ctgactcgct acgctcggtc
     2881 gttcgactgc ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc
     2941 aggaagatac ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc
     3001 gcccccctga caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag
     3061 gactataaag ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg
     3121 cctttcggtt taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg
     3181 acactcagtt ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt
     3241 cagtccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat
     3301 gcaaaagcac cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc
     3361 atgcgccggt taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca
     3421 gttacctcgg ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc
     3481 ggttttttcg ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca
     3541 tcttattaat cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc
     3601 acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc
     3661 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     3721 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3781 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3841 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     3901 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     3961 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4021 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4081 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     4141 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     4201 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     4261 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     4321 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     4381 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     4441 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     4501 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     4561 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     4621 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     4681 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     4741 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     4801 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     4861 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     4921 gatctcgacg ctctccctta tgcgactcct gcattaggaa attaatacga ctcactata
//
