pRGPDuo2 vector (V015847)

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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)."