LOCUS       40924_44998        9404 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Tn7 integrating plasmid with S. pasteurianus dCas9 expressed from 
            the Plac promoter for expression in P. aeruginosa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9404)
  AUTHORS   Tan SZ, Reisch CR, Prather KLJ
  TITLE     A Robust CRISPR Interference Gene Repression System in Pseudomonas.
  JOURNAL   J Bacteriol. 2018 Mar 12;200(7). pii: JB.00575-17. doi: 
            10.1128/JB.00575-17. Print 2018 Apr 1.
  PUBMED    29311279
REFERENCE   2  (bases 1 to 9404)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9404)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J
            Bacteriol."; date: "2018-03-12"; pages: " 10.1128/JB.00575-17. Print
            2018 Apr 1"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     100..119
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     534..550
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      578..672
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      775..861
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     protein_bind    907..954
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(1087..1617)
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(1806..1834)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    1875..1922
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(1968..3047)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(3048..3125)
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     promoter        3355..3385
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3393..3409
                     /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)."
     CDS             3461..3487
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     terminator      6981..7007
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     mobile_element  complement(7099..7264)
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      complement(7534..8122)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8296..9153)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(9154..9258)
                     /label=AmpR promoter
     primer_bind     9326..9344
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     primer_bind     complement(9382..9404)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
       61 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
      121 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
      181 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
      241 cttcgctatt acgccagcta gaggaccagc cgcgtaacct ggcaaaatcg gttacggttg
      301 agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa
      361 caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga
      421 aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact
      481 cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggg gttcgaggtc
      541 gacggtatcg ataagctagc ttaattagct gagcttggac tcctgttgat agatccagta
      601 atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt
      661 attggtgaga atccaagcta gactgcgatg agtggcaggg cggggcgtaa tttttttaag
      721 gcagttattg gtgcccttaa acgcctgggg taatgactct ctagcttgag gcatcaaata
      781 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
      841 gctctcctga gtaggacaaa tccgccgcta ggagcttgcg gcccggacga tcgaattggg
      901 gatcttgaag tacctattcc gaagttccta ttctctagaa agtataggaa cttcagagcg
      961 cttttgaagc tgatgtgctt aaaaacttac tcaatggaat tctagatcga attggccgcg
     1021 gcgttgtgac aatttaccga acaactccgc ggccgggaag ccgatctcgg cttgaacgaa
     1081 ttgttaggtg gcggtacttg ggtcgatatc aaagtgcatc acttcttccc gtatgcccaa
     1141 ctttgtatag agagccactg cgggatcgtc accgtaatct gcttgcacgt agatcacata
     1201 agcaccaagc gcgttggcct catgcttgag gagattgatg agcgcggtgg caatgccctg
     1261 cctccggtgc tcgccggaga ctgcgagatc atagatatag atctcactac gcggctgctc
     1321 aaacttgggc agaacgtaag ccgcgagagc gccaacaacc gcttcttggt cgaaggcagc
     1381 aagcgcgatg aatgtcttac tacggagcaa gttcccgagg taatcggagt ccggctgatg
     1441 ttgggagtag gtggctacgt ctccgaactc acgaccgaaa agatcaagag cagcccgcat
     1501 ggatttgact tggtcagggc cgagcctaca tgtgcgaatg atgcccatac ttgagccacc
     1561 taactttgtt ttagggcgac tgccctgctg cgtaacatcg ttgctgctgc gtaacatcgt
     1621 tgctgctcca taacatcaaa catcgaccca cggcgtaacg cgcttgctgc ttggatgccc
     1681 gaggcataga ctgtacaaaa aaacagtcat aacaagccat gaaaaccgcc actgcgccgt
     1741 taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata cgctacttgc
     1801 attacagttt acgaaccgaa caggcttatg tcaattcgat ccattgctgt tgacaaaggg
     1861 aatcagggga tcttgaagtt cctattccga agttcctatt ctctagaaag tataggaact
     1921 tcagagcgct tttgaagcta attcgatcat gcattgcgtt gcgctcactg cccgctttcc
     1981 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2041 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     2101 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     2161 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     2221 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     2281 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt aggaacgatg
     2341 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     2401 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     2461 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     2521 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     2581 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     2641 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     2701 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     2761 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     2821 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     2881 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     2941 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     3001 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     3061 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg
     3121 gtgtcaacgt aaatgccgct tcgccttcgc gcgcgaattg caagctgatc cgggcttatc
     3181 gactgcacgg tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct
     3241 gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata
     3301 atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgtttaca
     3361 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     3421 aaacagaatt cgagctcctg actcaggagg tatcgatatg tacccatacg atgttccaga
     3481 ttacgctatg accaacggca agatcctggg cctggccatc ggcatcgcct cggtgggcgt
     3541 gggcatcatc gaggccaaga ccggcaaggt ggtgcacgcc aactcgcgcc tgttctcggc
     3601 cgccaacgcc gagaacaacg ccgagcgccg cggcttccgc ggctcgcgcc gcctgaaccg
     3661 ccgcaagaag caccgcgtga agcgcgtgcg cgacctgttc gagaagtacg gcatcgtgac
     3721 cgacttccgc aacctgaacc tgaacccgta cgagctgcgc gtgaagggcc tgaccgagca
     3781 gctgaagaac gaggagctgt tcgccgccct gcgcaccatc tcgaagcgcc gcggcatctc
     3841 gtacctggac gacgccgagg acgactcgac cggctcgacc gactacgcca agtcgatcga
     3901 cgagaaccgc cgcctgctga agaacaagac cccgggccag atccagctgg agcgcctgga
     3961 gaagtacggc cagctgcgcg gcaacttcac cgtgtacgac gagaacggcg aggcccaccg
     4021 cctgatcaac gtgttctcga cctcggacta cgagaaggag gcccgcaaga tcctggagac
     4081 ccaggccgac tacaacaaga agatcaccgc cgagttcatc gacgactacg tggagatcct
     4141 gacccagaag cgcaagtact accacggccc gggcaacgag aagtcgcgca ccgactacgg
     4201 ccgcttccgc accgacggca ccaccctgga gaacatcttc ggcatcctga tcggcaagtg
     4261 caacttctac ccggacgagt accgcgcctc gaaggcctcg tacaccgccc aggagtacaa
     4321 cttcctgaac gacctgaaca acctgaaggt gtcgaccgag accggcaagc tgtcgaccga
     4381 gcagaaggag tcgctggtgg agttcgccaa gaacaccgcc accctgggcc cggccaagct
     4441 gctgaaggag atcgccaaga tcctggactg caaggtggac gagatcaagg gctaccgcga
     4501 ggacgacaag ggcaagccgg acctgcacac cttcgagccg taccgcaagc tgaagttcaa
     4561 cctggagtcg atcaacatcg acgacctgtc gcgcgaggtg atcgacaagc tggccgacat
     4621 cctgaccctg aacaccgagc gcgagggcat cgaggacgcc atcaagcgca acctgccgaa
     4681 ccagttcacc gaggagcaga tctcggagat catcaaggtg cgcaagtcgc agtcgaccgc
     4741 cttcaacaag ggctggcact cgttctcggc caagctgatg aacgagctga tcccggagct
     4801 gtacgccacc tcggacgagc agatgaccat cctgacccgc ctggagaagt tcaaggtgaa
     4861 caagaagtcg tcgaagaaca ccaagaccat cgacgagaag gaggtgaccg acgagatcta
     4921 caacccggtg gtggccaagt cggtgcgcca gaccatcaag atcatcaacg ccgccgtgaa
     4981 gaagtacggc gacttcgaca agatcgtgat cgagatgccg cgcgacaaga acgccgacga
     5041 cgagaagaag ttcatcgaca agcgcaacaa ggagaacaag aaggagaagg acgacgccct
     5101 gaagcgcgcc gcctacctgt acaactcgtc ggacaagctg ccggacgagg tgttccacgg
     5161 caacaagcag ctggagacca agatccgcct gtggtaccag cagggcgagc gctgcctgta
     5221 ctcgggcaag ccgatctcga tccaggagct ggtgcacaac tcgaacaact tcgagatcga
     5281 cgccatcctg ccgctgtcgc tgtcgttcga cgactcgctg gccaacaagg tgctggtgta
     5341 cgcctggacc aaccaggaga agggccaaaa gaccccgtac caggtgatcg actcgatgga
     5401 cgccgcctgg tcgttccgcg agatgaagga ctacgtgctg aagcagaagg gcctgggcaa
     5461 gaagaagcgc gactacctgc tgaccaccga gaacatcgac aagatcgagg tgaagaagaa
     5521 gttcatcgag cgcaacctgg tggacacccg ctacgcctcg cgcgtggtgc tgaactcgct
     5581 gcagtcggcc ctgcgcgagc tgggcaagga caccaaggtg tcggtggtgc gcggccagtt
     5641 cacctcgcag ctgcgccgca agtggaagat cgacaagtcg cgcgagacct accaccacca
     5701 cgccgtggac gccctgatca tcgccgcctc gtcgcagctg aagctgtggg agaagcagga
     5761 caacccgatg ttcgtggact acggcaagaa ccaggtggtg gacaagcaga ccggcgagat
     5821 cctgtcggtg tcggacgacg agtacaagga gctggtgttc cagccgccgt accagggctt
     5881 cgtgaacacc atctcgtcga agggcttcga ggacgagatc ctgttctcgt accaggtgga
     5941 ctcgaagtac aaccgcaagg tgtcggacgc caccatctac tcgacccgca aggccaagat
     6001 cggcaaggac aagaaggagg agacctacgt gctgggcaag atcaaggaca tctactcgca
     6061 gaacggcttc gacaccttca tcaagaagta caacaaggac aagacccagt tcctgatgta
     6121 ccagaaggac tcgctgacct gggagaacgt gatcgaggtg atcctgcgcg actacccgac
     6181 caccaagaag tcggaggacg gcaagaacga cgtgaagtgc aacccgttcg aggagtaccg
     6241 ccgcgagaac ggcctgatct gcaagtactc gaagaagggc aagggcaccc cgatcaagtc
     6301 gctgaagtac tacgacaaga agctgggcaa ctgcatcgac atcaccccgg aggagtcgcg
     6361 caacaaggtg atcctgcagt cgatcaaccc gtggcgcgcc gacgtgtact tcaacccgga
     6421 gaccctgaag tacgagctga tgggcctgaa gtactcggac ctgtcgttcg agaagggcac
     6481 cggcaactac cacatctcgc aggagaagta cgacgccatc aaggagaagg agggcatcgg
     6541 caagaagtcg gagttcaagt tcaccctgta ccgcaacgac ctgatcctga tcaaggacat
     6601 cgcctcgggc gagcaggaga tctaccgctt cctgtcgcgc accatgccga acgtgaacca
     6661 ctacgtggag ctgaagccgt acgacaagga gaagttcgac aacgtgcagg agctggtgga
     6721 ggccctgggc gaggccgaca aggtgggccg ctgcatcaag ggcctgaaca agccgaacat
     6781 ctcgatctac aaggtgcgca ccgacgtgct gggcaacaag tacttcgtga agaagaaggg
     6841 cgacaagccg aagctggact tcaagaacaa caagaagtaa atactctggc catttttctt
     6901 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcaacgcgtg
     6961 cggccgctga ttagctaagc agaaggccat cctgacggat gcctttttcg cttgctagct
     7021 gcagcccggg ggatcccccg ggctgcagga attcctcgag aagcttgggc ccggtacctc
     7081 gcgaaggcct tgcaggccaa ccagataagt gaaatctagt tccaaactat tttgtcattt
     7141 ttaattttcg tattagctta cgacgctaca cccagttccc atctattttg tcactcttcc
     7201 ctaaataatc cttaaaaact ccatttccac ccctcccagt tcccaactat tttgtccgcc
     7261 cacagcgggg catttttctt cctgttatgt ttgggcgctg cattaatgaa tcggccaacg
     7321 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgacccgct
     7381 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     7441 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     7501 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     7561 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     7621 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     7681 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     7741 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     7801 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     7861 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     7921 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     7981 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     8041 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     8101 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     8161 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     8221 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     8281 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     8341 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     8401 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     8461 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     8521 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     8581 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     8641 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     8701 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     8761 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     8821 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     8881 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     8941 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     9001 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     9061 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     9121 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     9181 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     9241 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     9301 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     9361 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccgg
//
