LOCUS       Exported                9952 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pBT20-Dbla-T7pol, complete sequence.
ACCESSION   EF153732
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9952)
  AUTHORS   Kang Y, Son MS, Hoang TT.
  TITLE     One step engineering of T7-expression strains for protein 
            production: increasing the host-range of the T7-expression system
  JOURNAL   Protein Expr. Purif. 55 (2), 325-333 (2007)
  PUBMED    17716915
REFERENCE   2  (bases 1 to 9952)
  AUTHORS   Kang Y, Son MS, Hoang TT.
  TITLE     Direct Submission
  JOURNAL   Submitted (30-NOV-2006) Microbiology, University of Hawaii at Manoa,
            2538 The Mall - Snyder 310, Honolulu, HI 96822, USA
REFERENCE   3  (bases 1 to 9952)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9952
                     /organism="Expression vector pBT20-Dbla-T7pol"
                     /mol_type="other DNA"
                     /db_xref="taxon:418828"
     CDS             34..405
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     rep_origin      1021..1409
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     rep_origin      complement(1029..1300)
                     /direction=LEFT
                     /label=R6K-gamma-ori
                     /note="R6K-gamma-ori"
     repeat_region   1451..1478
                     /rpt_family="Mariner"
     CDS             complement(1546..4197)
                     /codon_start=1
                     /gene="T7-pol"
                     /product="T7 RNA polymerase"
                     /label=T7-pol
                     /protein_id="ABN54772.1"
                     /translation="MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYE
                     MGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQ
                     FLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFK
                     KNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIE
                     STGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGI
                     TGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANV
                     ITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLE
                     FMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYY
                     WLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFE
                     YAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAK
                     KVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVM
                     TLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVE
                     AMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLG
                     QFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHD
                     SFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLN
                     LRDILESDFAFA"
     gene            complement(1546..4197)
                     /gene="T7-pol"
                     /label=T7-pol
     CDS             complement(4194..4679)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVRDLIPDLLTGRGTK"
     primer_bind     4643..4659
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4675..4691)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    complement(4697..4721)
                     /label=lac operator
                     /note="lac operator"
     protein_bind    4699..4715
                     /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(4723..4753)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     regulatory      4724..4753
                     /regulatory_class="promoter"
                     /note="lacUV5 promoter"
     protein_bind    4768..4789
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(4802..5884)
                     /codon_start=1
                     /gene="lacIq"
                     /product="LacI"
                     /label=lacIq
                     /note="lac repressor"
                     /protein_id="ABN54773.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(4802..5884)
                     /gene="lacIq"
                     /label=lacIq
     misc_feature    complement(5965..6012)
                     /note="FRT; Flp-recombination-target"
     protein_bind    complement(5965..6012)
                     /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)."
     promoter        6053..6081
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             6270..6803
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyl transferase"
                     /function="gentamycin resistance"
                     /label=aacC1
                     /protein_id="ABN54774.1"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     gene            6270..6803
                     /gene="aacC1"
                     /label=aacC1
     misc_feature    complement(6933..6980)
                     /note="FRT; Flp-recombination-target"
     protein_bind    complement(6933..6980)
                     /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             7243..7698
                     /codon_start=1
                     /gene="T7-lys"
                     /product="T7 lysozyme"
                     /label=T7-lys
                     /note="native inhibitor of T7 RNA polymerase"
                     /protein_id="ABN54775.1"
                     /translation="MARVQFKQRESTDAIFVHCSATKPSQNVGVREIRQWHKEQGWLDV
                     GYHFIIKRDGTVEAGRDEMAVGSHAKGYNHNSIGVCLVGGIDDKGKFDANFTPAQMQSL
                     RSLLVTLLAKYEGAGLRAHHEVAPKACPSFDLKRWWEKNELVTSDRG"
     gene            7243..7698
                     /gene="T7-lys"
                     /label=T7-lys
     repeat_region   7730..7757
                     /rpt_family="Mariner"
     primer_bind     7890..7906
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        7916..7934
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(7997..9043)
                     /codon_start=1
                     /gene="Tnp"
                     /product="Tnp"
                     /label=Tnp
                     /note="mariner transposase"
                     /protein_id="ABN54776.1"
                     /translation="MEKKEFRVLIKYCFLKGKNTVEAKTWLDNEFPDSAPGKSTIIDWY
                     AKFKRGEMSTEDGERSGRPKEVVTDENIKKIHKMILNDRKMKLIEIAEALKISKERVGH
                     IIHQYLDMRKLCAKWVPRELTFDQKQRRVDDSKRCLQLLTRNTPEFFRRYVTMDETWLH
                     HYTPESNRQSAEWTATGEPSPKRGKTQKSAGKVMASVFWDAHGIIFIDYLEKGKTINSD
                     YYMALLERLKVEIAAKRPHMKKKKVLFHQDNAPCHKSLRTMAKIHELGFELLPHPPYSP
                     DLAPSDFFLFSDLKRMLAGKKFGCNEEVIAETEAYFEAKPKEYYQNGIKKLEGRYNRCI
                     ALEGNYVE"
     gene            complement(7997..9043)
                     /gene="Tnp"
                     /label=Tnp
     misc_recomb     9711..9952
                     /note="oriT; conjugal origin of transfer"
     oriT            9844..1
                     /note="incP origin of transfer"