LOCUS       62056_18840        7928 bp DNA     circular SYN 20-JUN-2020
DEFINITION  Cloning vector pRGPDuo2, complete sequence.
ACCESSION   MN913428
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7928)
  AUTHORS   Gauttam R, Mukhopadhyay A, Singer SW.
  TITLE     Construction of a novel dual-inducible duet-expression system for 
            gene (over)expression in Pseudomonas putida
  JOURNAL   Plasmid, 102514 (2020) In press
  PUBMED    32504628
REFERENCE   2  (bases 1 to 7928)
  AUTHORS   Gauttam R, Singer SW.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-JAN-2020) Joint BioEnergy Institute, Lawrence Berkeley
            National Laboratory, 5885 Hollis Street, Emeryville, CA 94608, USA
REFERENCE   3  (bases 1 to 7928)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plasmid, 
            102514 (2020) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (08-JAN-2020) Joint BioEnergy Institute, Lawrence Berkeley National 
            Laboratory, 5885 Hollis Street, Emeryville, CA 94608, USA"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    complement(1..57)
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(58..74)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      287..373
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      465..492
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        511..602
                     /label=AmpR promoter
     primer_bind     complement(923..939)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(947..963)
                     /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)."
     promoter        complement(971..1001)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(1016..1037)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(1325..1913)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             2146..2958
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      complement(3719..3746)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(3748..3794)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     promoter        complement(3897..3952)
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     CDS             3968..4591
                     /codon_start=1
                     /label=TetR
                     /note="tetracycline repressor TetR"
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     promoter        complement(4701..4729)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      complement(4779..5130)
                     /direction=LEFT
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     CDS             5144..5974
                     /codon_start=1
                     /label=pRO1600 Rep
                     /note="replication protein for the broad-host-range plasmid
                     pRO1600 from Pseudomonas aeruginosa"
                     /translation="VASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     promoter        complement(6348..6376)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     CDS             complement(6491..7519)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ASA"
     promoter        complement(7520..7597)
                     /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        7831..7859
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    7867..7883
                     /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)."
ORIGIN
        1 aagcttgcat gcctgcaggt cgactctaga ggatccccgg gtaccgagct cgaattcact
       61 ggccgtcgtt ttacagccaa gcttggctgt tttggcggat gagagaagat tttcagcctg
      121 atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt
      181 agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat
      241 ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa
      301 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
      361 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
      421 gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac
      481 ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata
      541 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
      601 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
      661 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
      721 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
      781 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
      841 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
      901 tggttgagta attcgtaatc atgtcatagc tgtttcctgt gtgaaattgt tatccgctca
      961 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     1021 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     1081 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     1141 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     1201 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     1261 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     1321 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     1381 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     1441 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     1501 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     1561 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     1621 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     1681 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     1741 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     1801 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     1861 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     1921 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     1981 tggtcatgag attatcaaaa aggatcttca cctagatcct tttggggggg gggggaaagc
     2041 cacgttgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga
     2101 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     2161 cgggaaacgt cttgctcgag gccgcgatta aattccaaca tggatgctga tttatatggg
     2221 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg
     2281 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     2341 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     2401 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca
     2461 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     2521 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     2581 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     2641 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataagctt
     2701 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     2761 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     2821 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     2881 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     2941 atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt
     3001 acgctgactt gacgggacgg cggctttgtt gaataaatcg aacttttgct gagttgaagg
     3061 atcagatcac gcatcttccc gacaacgcag accgttccgt ggcaaagcaa aagttcaaaa
     3121 tcaccaactg gtccacctac aacaaagctc tcatcaaccg tggctccctc actttctggc
     3181 tggatgatgg ggcgattcag gcctggtatg agtcagcaac accttcttca cgaggcagac
     3241 ctcagcgccc ccccccccct agcttgtcta cgtctgatgc tttgaatcgg acggacttgc
     3301 cgatcttgta tgcggtgatt tttccctcgt ttgcccactt tttaatggtg gccggggtga
     3361 gagctacgcg ggcggcgacc tgctgcgctg tgatccaata ttcggggtcg ttcactggtt
     3421 cccctttctg atttctggca tagaagaacc cccgtgaact gtgtggttcc gggggttgct
     3481 gatttttgcg agacttctcg cgcaattccc tagcttaggt gaaaacacca tgaaacacta
     3541 gggaaacacc catgaaacac ccattagggc agtagggcgg cttcttcgtc tagggcttgc
     3601 atttgggcgg tgatctggtc tttagcgtgt gaaagtgtgt cgtaggtggc gtgctcaatg
     3661 cactcgaacg tcacgtcatt taccgggtca cggtgggcaa agagaactag tttcgaaacg
     3721 cagaaaggcc cacccgaagg tgagccaata aaacgaaagg cccagtcttt cgactgagcc
     3781 tttcgtttta tttgatgcct ggaccgcggt actccgcggg ctgaggacat gttgtacaat
     3841 ttaaatccta gggctagcgt ataccctttc ctcctcttta gatcttttga attcttttct
     3901 ctatcactga tagggagtgg taaaataact ctatcaacga tagagtgtca acaaaaatta
     3961 ggaattaatg atgtctagat tagataaaag taaagtgatt aacagcgcat tagagctgct
     4021 taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga
     4081 gcagcctaca ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat
     4141 tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga
     4201 ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc
     4261 aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt
     4321 agccttttta tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt
     4381 ggggcatttt actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga
     4441 aagggaaaca cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt
     4501 tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt
     4561 agaaaaacaa cttaaatgtg aaagtgggtc ttaagacgtc gatcaattga gccatgaaaa
     4621 ccgccactgc gccgttacca ccgctgcgtt cggtcaaggt tctggaccag ttgcgtgagc
     4681 gcatacgcta cttgcattac agtttacgaa ccgaacaggc ttatgtcaat tcgcctctca
     4741 ggcgccgctg gtgccgctgg ttggacgcca agggtgaatc cgcctcgata ccctgattac
     4801 tcgcttcctg cgccctctca ggcggcgata ggggactggt aaaacgggga ttgcccagac
     4861 gcctcccccg ccccttcagg ggcacaaatg cggccccaac ggggccacgt agtggtgcgt
     4921 tttttgcgtt tccacccttt tcttcctttt cccttttaaa ccttttagga cgtctacagg
     4981 ccacgtaatc cgtggcctgt agagtttaaa aagggacgga tttgttgcca ttaagggacg
     5041 gatttgttgt taagaaggga cggatttgtt gttgtaaagg gacggatttg ttgtattgtg
     5101 ggacgcagat acagtgtccc cttatacaca aggaatgtcg aacgtggcct cacccccaat
     5161 ggtttacaaa agcaatgccc tggtcgaggc cgcgtatcgc ctcagtgttc aggaacagcg
     5221 gatcgttctg gcctgtatta gccaggtgaa gaggagcgag cctgtcaccg atgaagtgat
     5281 gtattcagtg acggcggagg acatagcgac gatggcgggt gtccctatcg aatcttccta
     5341 caaccagctc aaagaagcgg ccctgcgcct gaaacggcgg gaagtccggt taacccaaga
     5401 gcccaatggc aaggggaaaa gaccgagtgt gatgattacc ggctgggtgc aaacaatcat
     5461 ctaccgggag ggtgagggcc gtgtagaact caggttcacc aaagacatgc tgccgtacct
     5521 gacggaactc accaaacagt tcaccaaata cgccttggct gacgtggcca agatggacag
     5581 cacccacgcg atcaggcttt acgagctgct catgcaatgg gacagcatcg gccagcgcga
     5641 aatagaaatt gaccagctgc gaaagtggtt tcaactggaa ggccggtatc cctcgatcaa
     5701 ggacttcaag ttgcgagtgc ttgatccagc cgtgacgcag atcaacgagc acagcccgct
     5761 acaggtggag tgggcgcagc gaaagaccgg gcgcaaggtc acacatctgt tgttcagttt
     5821 tggaccgaag aagcccgcca aggcggtggg taaggcccca gcgaagcgca aggccgggaa
     5881 gatttcagat gctgagatcg cgaaacaggc tcgccctggt gagacatggg aagcggcccg
     5941 cgctcgacta acccagatgc cgctggatct ggcctagagg ccgtggccac cacggcccgg
     6001 cctgcctttc aggctgcgca actgcgtacg aggcccggcg ccagcgcgag gcgatcgtgt
     6061 ccgccgtggc gtcggcagtc gctcgccgag atacgtcacg tgacgaaatg cagcagcctt
     6121 ccattccgtc acgtgacgaa actcgggccg caggtcagag cacggttccg cccgctccgg
     6181 ccctgccgga cccccggcat cccgcaagag gcccggcagt accggcataa ccaagcctat
     6241 gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat
     6301 acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc
     6361 gatgataagc tgtcaaacat ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg
     6421 ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg
     6481 ccggcatggc ggccgacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     6541 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     6601 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     6661 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     6721 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     6781 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     6841 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     6901 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     6961 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     7021 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     7081 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     7141 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     7201 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     7261 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     7321 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     7381 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     7441 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     7501 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     7561 ccataccgcg aaaggttttg caccattcga tggtgtcaac gtaaatgcat gccgcttcgc
     7621 cttcgcgcgc gaattgcaag ctgatccggg cttatcgact gcacggtgca ccaatgcttc
     7681 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata
     7741 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa
     7801 cggttctggc aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt
     7861 gtggaattgt gagcggataa caatttcaca caggaaacag aattaaaaga tatgaccatg
     7921 attacgcc
//
