LOCUS       62056_18845        8549 bp DNA     circular SYN 15-NOV-2020
DEFINITION  Cloning vector pRGPDuo4, complete sequence.
ACCESSION   MT304467
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8549)
  AUTHORS   Gauttam R, Singer SW.
  TITLE     Pseudomonas 2.0: Upgradable next-generation compatible 
            duet-expression vectors for metabolic engineering research in 
            Pseudomonas putida
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8549)
  AUTHORS   Gauttam R, Singer SW.
  TITLE     Direct Submission
  JOURNAL   Submitted (07-APR-2020) Joint BioEnergy Institute, Lawrence Berkeley
            National Laboratory, 5885 Hollis Street, Emeryville, CA 94608, USA
REFERENCE   3  (bases 1 to 8549)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (07-APR-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..8549
                     /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(3927..4211)
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     CDS             4238..5113
                     /codon_start=1
                     /label=araC
                     /note="L-arabinose regulatory protein"
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        complement(5322..5350)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      complement(5400..5751)
                     /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             5765..6595
                     /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(6969..6997)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     CDS             complement(7112..8140)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ASA"
     promoter        complement(8141..8218)
                     /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        8452..8480
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    8488..8504
                     /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 gggctagcgc ggccgcacca tggagatctt ttgaattcca aaattatttc
     3901 tagagggaaa ccgttgtggt ctccctatgg agaaacagta gagagttgcg ataaaaagcg
     3961 tcaggtagaa tccgctaatc ttatggataa aaatgctatg gcatagcaaa gtgtgacgcc
     4021 gtgcaaataa tcaatgtgga cttttctgcc gtgattatag acacttttgt tacgcgtttt
     4081 tgtcatggct ttggtcccgc tttgttacag aatgctttta ataagcgggg ttaccggttt
     4141 ggttagcgag aagagccagt aaaagacgca gtgacggcaa tgtctgatgc aatatggaca
     4201 attggtttct tctctgaatg gcgggagtat gaaaagtatg gctgaagcgc aaaatgatcc
     4261 cctgctgccg ggatactcgt ttaatgccca tctggtggcg ggtttaacgc cgattgaggc
     4321 caacggttat ctcgattttt ttatcgaccg accgctggga atgaaaggtt atattctcaa
     4381 tctcaccatt cgcggtcagg gggtggtgaa aaatcaggga cgagaatttg tttgccgacc
     4441 gggtgatatt ttgctgttcc cgccaggaga gattcatcac tacggtcgtc atccggaggc
     4501 tcgcgaatgg tatcaccagt gggtttactt tcgtccgcgc gcctactggc atgaatggct
     4561 taactggccg tcaatatttg ccaatacggg gttctttcgc ccggatgaag cgcaccagcc
     4621 gcatttcagc gacctgtttg ggcaaatcat taacgccggg caaggggaag ggcgctattc
     4681 ggagctgctg gcgataaatc tgcttgagca attgttactg cggcgcatgg aagcgattaa
     4741 cgagtcgctc catccaccga tggataatcg ggtacgcgag gcttgtcagt acatcagcga
     4801 tcacctggca gacagcaatt ttgatatcgc cagcgtcgca cagcatgttt gcttgtcgcc
     4861 gtcgcgtctg tcacatcttt tccgccagca gttagggatt agcgtcttaa gctggcgcga
     4921 ggaccaacgt atcagccagg cgaagctgct tttgagcacc acccggatgc ctatcgccac
     4981 cgtcggtcgc aatgttggtt ttgacgatca actctatttc tcgcgggtat ttaaaaaatg
     5041 caccggggcc agcccgagcg agttccgtgc cggttgtgaa gaaaaagtga atgatgtagc
     5101 cgtcaagttg tcataagacg tcggaattgc cagctggggc gccctctggt aaggttggga
     5161 agccctgcac atatgcggat tagaaaaaca acttaaatgt gaaagtgggt cttaagacgt
     5221 cgatcaattg agccatgaaa accgccactg cgccgttacc accgctgcgt tcggtcaagg
     5281 ttctggacca gttgcgtgag cgcatacgct acttgcatta cagtttacga accgaacagg
     5341 cttatgtcaa ttcgcctctc aggcgccgct ggtgccgctg gttggacgcc aagggtgaat
     5401 ccgcctcgat accctgatta ctcgcttcct gcgccctctc aggcggcgat aggggactgg
     5461 taaaacgggg attgcccaga cgcctccccc gccccttcag gggcacaaat gcggccccaa
     5521 cggggccacg tagtggtgcg ttttttgcgt ttccaccctt ttcttccttt tcccttttaa
     5581 accttttagg acgtctacag gccacgtaat ccgtggcctg tagagtttaa aaagggacgg
     5641 atttgttgcc attaagggac ggatttgttg ttaagaaggg acggatttgt tgttgtaaag
     5701 ggacggattt gttgtattgt gggacgcaga tacagtgtcc ccttatacac aaggaatgtc
     5761 gaacgtggcc tcacccccaa tggtttacaa aagcaatgcc ctggtcgagg ccgcgtatcg
     5821 cctcagtgtt caggaacagc ggatcgttct ggcctgtatt agccaggtga agaggagcga
     5881 gcctgtcacc gatgaagtga tgtattcagt gacggcggag gacatagcga cgatggcggg
     5941 tgtccctatc gaatcttcct acaaccagct caaagaagcg gccctgcgcc tgaaacggcg
     6001 ggaagtccgg ttaacccaag agcccaatgg caaggggaaa agaccgagtg tgatgattac
     6061 cggctgggtg caaacaatca tctaccggga gggtgagggc cgtgtagaac tcaggttcac
     6121 caaagacatg ctgccgtacc tgacggaact caccaaacag ttcaccaaat acgccttggc
     6181 tgacgtggcc aagatggaca gcacccacgc gatcaggctt tacgagctgc tcatgcaatg
     6241 ggacagcatc ggccagcgcg aaatagaaat tgaccagctg cgaaagtggt ttcaactgga
     6301 aggccggtat ccctcgatca aggacttcaa gttgcgagtg cttgatccag ccgtgacgca
     6361 gatcaacgag cacagcccgc tacaggtgga gtgggcgcag cgaaagaccg ggcgcaaggt
     6421 cacacatctg ttgttcagtt ttggaccgaa gaagcccgcc aaggcggtgg gtaaggcccc
     6481 agcgaagcgc aaggccggga agatttcaga tgctgagatc gcgaaacagg ctcgccctgg
     6541 tgagacatgg gaagcggccc gcgctcgact aacccagatg ccgctggatc tggcctagag
     6601 gccgtggcca ccacggcccg gcctgccttt caggctgcgc aactgcgtac gaggcccggc
     6661 gccagcgcga ggcgatcgtg tccgccgtgg cgtcggcagt cgctcgccga gatacgtcac
     6721 gtgacgaaat gcagcagcct tccattccgt cacgtgacga aactcgggcc gcaggtcaga
     6781 gcacggttcc gcccgctccg gccctgccgg acccccggca tcccgcaaga ggcccggcag
     6841 taccggcata accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat
     6901 gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa
     6961 actaccgcat taaagcttat cgatgataag ctgtcaaaca tggcctgtcg cttgcggtat
     7021 tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg
     7081 gcgagaagca ggccattatc gccggcatgg cggccgacgc gcggggagag gcggtttgcg
     7141 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     7201 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     7261 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     7321 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     7381 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     7441 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     7501 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     7561 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     7621 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     7681 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     7741 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     7801 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     7861 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     7921 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     7981 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     8041 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     8101 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     8161 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcaccattcg atggtgtcaa
     8221 cgtaaatgca tgccgcttcg ccttcgcgcg cgaattgcaa gctgatccgg gcttatcgac
     8281 tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg
     8341 caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg
     8401 ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct gttgacaatt
     8461 aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac acaggaaaca
     8521 gaattaaaag atatgaccat gattacgcc
//
