LOCUS       Exported                6972 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Bicistronic shuttle vector pDUO-26T, complete sequence.
ACCESSION   KX806599
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6972)
  AUTHORS   Nakata PA.
  TITLE     Construction of pDUO: A bicistronic shuttle vector series for dual 
            expression of recombinant proteins
  JOURNAL   Plasmid 89, 16-21 (2016)
  PUBMED    27989736
REFERENCE   2  (bases 1 to 6972)
  AUTHORS   Nakata PA.
  TITLE     Direct Submission
  JOURNAL   Submitted (31-AUG-2016) Pediatrics, USDA/ARS Childrens Nutrition 
            Research Center, Baylor College of Medicine, 1100 Bates Street, 
            Houston, TX 77030-2600, USA
REFERENCE   3  (bases 1 to 6972)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..6972
                     /organism="Bicistronic shuttle vector pDUO-26T"
                     /mol_type="other DNA"
                     /db_xref="taxon:1897403"
     promoter        complement(12..30)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(48..64)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    72..88
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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(96..126)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    141..162
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        409..437
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             485..1675
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline resistance marker"
                     /label=tet
                     /note="tetracycline efflux protein"
                     /protein_id="APR64513.1"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     gene            485..1675
                     /gene="tet"
                     /label=tet
     rep_origin      complement(2481..3100)
                     /direction=LEFT
                     /label=ori_pBR322
                     /note="ori_pBR322"
     rep_origin      2497..3085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     oriT            3150..3417
     oriT            3157..3265
                     /note="incP origin of transfer"
     CDS             complement(3422..4300)
                     /codon_start=1
                     /gene="araC"
                     /product="AraC"
                     /label=araC
                     /note="arabinose operon regulator"
                     /protein_id="APR64514.1"
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     gene            complement(3422..4300)
                     /gene="araC"
                     /label=araC
     promoter        4327..4611
                     /gene="araBAD"
                     /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)"
     misc_feature    4330..4345
                     /label=O2 region
                     /note="O2 region"
     misc_feature    4487..4508
                     /label=O1 region
                     /note="O1 region"
     misc_feature    4529..4542
                     /label=CAP binding site
                     /note="CAP binding site"
     misc_feature    4539..4577
                     /label=I2 and I1 region
                     /note="I2 and I1 region"
     RBS             4638..4660
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     misc_feature    4654..4659
                     /label=RBS
                     /note="RBS"
     misc_feature    4671..4762
                     /label=multiple cloning site
                     /note="multiple cloning site"
     misc_feature    4684..4701
                     /label=HIS tag
                     /note="HIS tag"
     CDS             4684..4701
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      4826..4983
                     /regulatory_class="terminator"
                     /note="rrnB T1 and T2 trancription terminator"
     terminator      4946..4992
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        4998..5282
                     /gene="araBAD"
                     /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)"
     misc_feature    5001..5016
                     /label=O2 region
                     /note="O2 region"
     misc_feature    5158..5179
                     /label=O1 region
                     /note="O1 region"
     misc_feature    5200..5213
                     /label=CAP binding site
                     /note="CAP binding site"
     misc_feature    5210..5248
                     /label=I2 and I1 region
                     /note="I2 and I1 region"
     RBS             5309..5331
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     misc_feature    5325..5330
                     /label=RBS
                     /note="RBS"
     misc_feature    5347..5482
                     /label=multiple cloning site
                     /note="multiple cloning site"
     misc_feature    5414..5458
                     /label=S tag
                     /note="S tag"
     CDS             5414..5458
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     regulatory      5537..5694
                     /regulatory_class="terminator"
                     /note="rrnB T1 and T2 trancription terminator"
     terminator      5657..5703
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5729..6571)
                     /codon_start=1
                     /gene="rep"
                     /product="Rep"
                     /label=rep
                     /note="replication protein"
                     /protein_id="APR64515.1"
                     /translation="MSNVASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVT
                     DEVMYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGW
                     VQTIIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWD
                     SIGQREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKV
                     THLLFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     gene            complement(5729..6571)
                     /gene="rep"
                     /label=rep
     CDS             complement(5729..6562)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pRO1600 from Pseudomonas aeruginosa "
                     /label=pRO1600 Rep
                     /translation="MASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     rep_origin      complement(6576..6972)
                     /direction=LEFT
                     /label=ori_pRO1614
                     /note="ori_pRO1614"
     rep_origin      6576..6927
                     /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)"