LOCUS       40924_40607        6858 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses V. cholerae TnsA, TnsB, and TnsC from a T7 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6858)
  AUTHORS   Klompe SE, Vo PLH, Halpin-Healy TS, Sternberg SH
  TITLE     Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA 
            integration.
  JOURNAL   Nature. 2019 Jul;571(7764):219-225. doi: 10.1038/s41586-019-1323-z. 
            Epub 2019 Jun 12.
  PUBMED    31189177
REFERENCE   2  (bases 1 to 6858)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6858)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-019-1323-z"; journalName: "Nature"; date: "2019-07";
            volume: "571"; issue: "7764"; pages: "219-225"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(164..185)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(201..1280)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(1281..1358)
                     /label=lacI promoter
     promoter        1404..1422
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1423..1447
                     /label=lac operator
                     /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             1462..1484
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3569..3580
                     /codon_start=1
                     /label=Factor Xa site
                     /note="Factor Xa recognition and cleavage site"
                     /translation="IEGR"
     terminator      5021..5068
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5301..6113)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(6114..6205)
                     /label=AmpR promoter
     rep_origin      complement(6223..6858)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
ORIGIN
        1 tcactctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata
       61 cgatataagt tgtaattctc atgttagtca tgccccgcgc ccaccggaag gagctgactg
      121 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
      181 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
      241 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
      301 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
      361 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
      421 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg
      481 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
      541 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
      601 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
      661 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
      721 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
      781 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
      841 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
      901 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
      961 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     1021 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     1081 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     1141 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     1201 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     1261 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     1321 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     1381 atgcgactcc tgcattagga aattaatacg actcactata ggggaattgt gagcggataa
     1441 caattcccct gtagaaataa ttttgtttaa ctttaataag gagatatacc atggcgacaa
     1501 gtttacctac gccctcagca attacgactt cggcgttaga gtatgcattc catactcccg
     1561 ctcgcaatct aacgaaatct cgcggaaaaa acattcatcg ttatgtcagt gtaaagatga
     1621 gtaagaggat tacggtagaa tctactctag agtgtgatgc ctgctatcac tttgattttg
     1681 agccaagtat tgttcgcttt tgcgctcaac cgattcgatt tttatattat ctcaatggtc
     1741 agtctcactc ctatgttcct gactttctag ttcaatttga taccaacgag tttgttctat
     1801 atgaagtaaa gtcagcttat gctaagaaca aacctgattt tgatgttgaa tgggaggcga
     1861 aagtaaaagc agcaactgaa ctagggctag aattggagct tgttgaagag agtgatatta
     1921 gggatacggt tgtattaaat aatcttaagc gcatgcatcg ttatgcttcg aaagatgagc
     1981 tgaataacgt acataactct ctcttaaaaa taataaagta caatggcgcc caatctgcaa
     2041 gatgcttggg agaacagttg ggtttaaaag gccgaactgt tttaccaatt ttgtgcgatt
     2101 tgctgtcaag gtgtttactc gatacacgtt tggataagcc tctatctctt gaatctcgat
     2161 ttgagttggc cagttatggc taagaaaggg ttctcaagtt tccatagaaa agcagtctct
     2221 tcccaagata cgcttgaatc gatagagctt gtctctagcg ctaattgtct agaaagtgtt
     2281 acgtatcaag atatatcagc atttcccgaa acaattgcgg tagagattaa tttccgatta
     2341 agcattcttc gctttttagc gcgaaagtgc gaaaccattg tggccaaatc aattgaacca
     2401 catcgtgtag agctacagca aaactatagt agaaaaatac ccagtgcaat aacgatatat
     2461 cgatggtggc ttgcttttcg aaaatcagac tacaacccca ttagcttagc acctaatatc
     2521 aaggatagag gtaatagaga gacaaaagtg tcaacagttg ttgattctat tatggaacag
     2581 gcagttgaaa gagttatatc tggacgaaaa gtcaatgtta gctctgcata taaacgtgtt
     2641 cgacgaaaag ttcgtcaata caatctaact catggaacga aatacacgta tcctaagtac
     2701 gaatctgtaa gaaagcgagt aaaaaagaaa accccatttg agttattagc cgcagggaaa
     2761 ggggagagag tagctaagag agagtttcgc cgaatgggaa aaaagatcct cacgtctagc
     2821 gtgctagaga gggttgaaat agatcacact gtcgttgacc tttttgcagt acacgaagag
     2881 tatcgaatcc cattgggccg accttggctt actcaattgg ttgattgtta cagtaaagct
     2941 gtaatcggtt tttatttagg tttcgagcct cctagctatg tgtcggtttc ccttgcactt
     3001 aagaatgcaa tacaacgcaa agatgactta atctcctcgt atgaatcgat cgagaatgaa
     3061 tggctatgtt atggcatccc agacctactc gtaactgata atggtaaaga gtttttgtcg
     3121 aaagcatttg atcaagcatg tgaatcacta ttgatcaatg tgcatcaaaa taaagttgag
     3181 acgcccgaca acaaacctca tgttgaacgt aactacggga ctattaatac ttctctgtta
     3241 gacgatttac ctgggaaatc cttcagccag taccttcaaa gagaagggta cgactctgtg
     3301 ggagaagcta cccttacact caatgagatt agagaaattt acttaatttg gttggtggat
     3361 atttatcata aaaaacccaa tcagagaggc actaattgtc ctaatgttgc gtggaaaaag
     3421 ggttgtcaag aatgggaacc agaggagttc tctggttcta aagacgaatt agactttaaa
     3481 tttgctattg ttgattacaa acaacttact aaagtaggga taactgtcta caaagaactg
     3541 agttatagca atgaccgttt agctgaatat agagggaaga aaggaaacca taaagttcag
     3601 ttcaagtata accctgagtg tatggcagtt atttgggtgt tggatgagga tatgaatgag
     3661 tactttacag ttaatgcgat tgactacgaa tatgcaagta gagtatcact ttggcaacat
     3721 aaatataaca tgaaatatca agcagaacta aattcagcag aatatgatga ggacaaggaa
     3781 attgatgcag aaataaaaat tgaagaaatc gcagatcgtt caattgttaa gactaacaaa
     3841 atcagagctc ggaggcgtgg cgctaggcat caagagaata gcgcaagggc taagtcaatc
     3901 agtaatgcga acccggcctc gatacaaaaa catgaagatg aaatcgttag tgcagataat
     3961 gacgattggg atattgatta tgtctgagaa atcgtcaaat gagtgaaacg cgtgaggctc
     4021 gtatatcaag agctaaaagg gcatttgtat ccacaccgag cgttaggaaa atcttgagtt
     4081 acatggatag atgtagagat ctatcagacc tagagtctga gcctacatgc atgatggtct
     4141 atggtgcttc gggtgtaggt aaaacgaccg tcatcaagaa atacttaaat caaaacagaa
     4201 gagagtccga agccgggggc gatataatac cggttttgca tattgagttg ccagacaatg
     4261 cgaagccagt agatgcagca agggaattgc tggttgaaat gggtgacccg ctagcacttt
     4321 atgaaactga cttagctaga ttgacgaaaa gactgactga attaatccct gcggtcggcg
     4381 tgaagctgat tattatcgat gagttccaac atttggtgga agaaaggtca aatcgggttc
     4441 ttacccaagt aggtaattgg ctaaaaatga tacttaacaa aacgaaatgt ccaattgtta
     4501 tatttggtat gccatactca aaagttgtac tgcaagcaaa ctcgcaactt cacgggcgat
     4561 tttccattca ggttgaactg cgccccttta gctaccaggg aggtagaggt gtatttaaaa
     4621 cttttttgga ataccttgat aaagccctac cttttgaaaa acaggctggc ttagccaacg
     4681 aaagtttgca gaaaaaattg tatgcattct ctcagggaaa catgcgttcg ttgagaaacc
     4741 ttatttatca agcatctatc gaagcaattg ataatcagca tgagacgata accgaagaag
     4801 atttcgtttt tgcatcgaag ttgacatcgg gcgataaacc caactcatgg aaaaatcctt
     4861 ttgaggaggg tgttgaggta acagaagata tgttacgacc gccaccaaaa gatattggtt
     4921 gggaggacta tttgagacat tcaaccccga gagtgagtaa accaggtaga aataaaaact
     4981 ttttcgaata acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     5041 ctctaaacgg gtcttgaggg gttttttgct gaaacctcag gcatttgaga agcacacggt
     5101 cacactgctt ccggtagtca ataaaccggt aaaccagcaa tagacataag cggctattta
     5161 acgaccctgc cctgaaccga cgacaagctg acgaccgggt ctccgcaagt ggcacttttc
     5221 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5281 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     5341 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     5401 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     5461 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     5521 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     5581 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     5641 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc ggtcgctgtt aaaaggacaa
     5701 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     5761 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     5821 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     5881 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     5941 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     6001 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     6061 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct catactcttc
     6121 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6181 gaatgtattt agaaaaataa acaaataggc atgctagcgc agaaacgtcc tagaagatgc
     6241 caggaggata cttagcagag agacaataag gccggagcga agccgttttt ccataggctc
     6301 cgcccccctg acgaacatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca
     6361 ggactataaa gataccaggc gtttccccct gatggctccc tcttgcgctc tcctgttccc
     6421 gtcctgcggc gtccgtgttg tggtggaggc tttacccaaa tcaccacgtc ccgttccgtg
     6481 tagacagttc gctccaagct gggctgtgtg caagaacccc ccgttcagcc cgactgctgc
     6541 gccttatccg gtaactatca tcttgagtcc aacccggaaa gacacgacaa aacgccactg
     6601 gcagcagcca ttggtaactg agaattagtg gatttagata tcgagagtct tgaagtggtg
     6661 gcctaacaga ggctacactg aaaggacagt atttggtatc tgcgctccac taaagccagt
     6721 taccaggtta agcagttccc caactgactt aaccttcgat caaaccgcct ccccaggcgg
     6781 ttttttcgtt tacagagcag gagattacga cgatcgtaaa aggatctcaa gaagatcctt
     6841 tacggattcc cgacacca
//
