LOCUS       Exported               10504 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Conjugative cloning vector pFE604T DNA, complete sequence.
ACCESSION   AB848355
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10504)
  AUTHORS   Yong HT, Yamamoto N, Takeuchi R, Hsieh YJ, Conrad TM, Datsenko KA, 
            Nakayashiki T, Wanner BL, Mori H.
  TITLE     Development of a System for Discovery of Genetic Interactions for 
            Essential Genes in Escherichia coli K-12
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 10504)
  AUTHORS   Mori H, Yong HT.
  TITLE     Direct Submission
  JOURNAL   Submitted (24-AUG-2013) Contact:Hirotada Mori Nara Institute of 
            Science and Technology, Graduate School of Biological Sciences; 
            8916-5 Takayama, Ikoma, Nara 630-0101, Japan URL 
            :http://ecoli.naist.jp/Lab
REFERENCE   3  (bases 1 to 10504)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10504
                     /organism="Conjugative cloning vector pFE604T"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /db_xref="taxon:1392254"
     promoter        24..67
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    44..60
                     /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)."
     protein_bind    74..93
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     RBS             110..121
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      502..596
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        933..961
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             1131..1664
                     /codon_start=1
                     /gene="gen"
                     /product="gentamicin-3-acetyltransferase"
                     /label=gen
                     /protein_id="BAN79343.1"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     gene            1131..1664
                     /gene="gen"
                     /label=gen
     oriT            1684..2018
     rep_origin      2037..2425
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     rep_origin      2075..2403
                     /label=oriRg, replication origin of R6K
                     /note="oriRg, replication origin of R6K"
     CDS             complement(2486..3112)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracyclin resistance regulator"
                     /label=tetR
                     /protein_id="BAN79344.1"
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     gene            complement(2486..3112)
                     /gene="tetR"
                     /label=tetR
     CDS             complement(2486..3109)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     promoter        3128..3183
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    3134..3152
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    3164..3182
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             3191..4396
                     /codon_start=1
                     /gene="tetA"
                     /product="tetracyclin resistance"
                     /label=tetA
                     /protein_id="BAN79345.1"
                     /translation="MNSSTKIALVITLLDAMGIGLIMPVLPTLLREFIASEDIANHFGV
                     LLALYALMQVIFAPWLGKMSDRFGRRPVLLLSLIGASLDYLLLAFSSALWMLYLGRLLS
                     GITGATGAVAASVIADTTSASQRVKWFGWLGASFGLGLIAGPIIGGFAGEISPHSPFFI
                     AALLNIVTFLVVMFWFRETKNTRDNTDTEVGVETQSNSVYITLFKTMPILLIIYFSAQL
                     IGQIPATVWVLFTENRFGWNSMMVGFSLAGLGLLHSVFQAFVAGRIATKWGEKTAVLLE
                     FIADSSAFAFLAFISEGWLDFPVLILLAGGGIALPALQGVMSIQTKSHEQGALQGLLVS
                     LTNATGVIGPLLFTVIYNHSLPIWDGWIWIIGLAFYCIIILLSMTFMLTPQAQGSKQET
                     SA"
     gene            3191..4396
                     /gene="tetA"
                     /label=tetA
     repeat_region   4807..5324
                     /gene="sopC"
     gene            4807..5324
                     /gene="sopC"
                     /label=sopC
     misc_feature    4851..5324
                     /gene="sopC"
                     /label=sopC
                     /note="centromere-like partitioning region of the bacterial
                     F plasmid"
     CDS             complement(5397..6368)
                     /codon_start=1
                     /gene="sopB"
                     /product="SopB protein"
                     /label=sopB
                     /protein_id="BAN79346.1"
                     /translation="MKRAPVIPKHTLNTQPVEDTSLSTPAAPMVDSLIARVGVMARGNA
                     ITLPVCGRDVKFTLEVLRGDSVEKTSRVWSGNERDQELLTEDALDDLIPSFLLTGQQTP
                     AFGRRVSGVIEIADGSRRRKAAALTESDYRVLVGELDDEQMAALSRLGNDYRPTSAYER
                     GQRYASRLQNEFAGNISALADAENISRKIITRCINTAKLPKSVVALFSHPGELSARSGD
                     ALQKAFTDKEELLKQQASNLHEQKKAGVIFEAEEVITLLTSVLKTSSASRTSLSSRHQF
                     APGATVLYKGDKMVLNLDRSRVPTECIEKIEAILKELEKPAP"
     gene            complement(5397..6368)
                     /gene="sopB"
                     /label=sopB
     CDS             complement(6368..7543)
                     /codon_start=1
                     /gene="sopA"
                     /product="SopA protein"
                     /label=sopA
                     /protein_id="BAN79347.1"
                     /translation="MFRMKLMETLNQCINAGHEMTKAIAIAQFNDDSPEARKITRRWRI
                     GEAADLVGVSSQAIRDAEKAGRLPHPDMEIRGRVEQRVGYTIEQINHMRDVFGTRLRRA
                     EDVFPPVIGVAAHKGGVYKTSVSVHLAQDLALKGLRVLLVEGNDPQGTASMYHGWVPDL
                     HIHAEDTLLPFYLGEKDDVTYAIKPTCWPGLDIIPSCLALHRIETELMGKFDEGKLPTD
                     PHLMLRLAIETVAHDYDVIVIDSAPNLGIGTINVVCAADVLIVPTPAELFDYTSALQFF
                     DMLRDLLKNVDLKGFEPDVRILLTKYSNSNGSQSPWMEEQIRDAWGSMVLKNVVRETDE
                     VGKGQIRMRTVFEQAIDQRSSTGAWRNALSIWEPVCNEIFDRLIKPRWEIR"
     gene            complement(6368..7543)
                     /gene="sopA"
                     /label=sopA
     misc_feature    7869..8119
                     /gene="incC"
                     /label=incC
                     /note="incompatibility region of the bacterial F plasmid"
     misc_feature    7872..8119
                     /label=incC
                     /note="incC"
     CDS             complement(8122..8877)
                     /codon_start=1
                     /gene="repE"
                     /product="replication initiation protein"
                     /label=repE
                     /protein_id="BAN79348.1"
                     /translation="MAETAVINHKKRKNSPRIVQSNDLTEAAYSLSRDQKRMLYLFVDQ
                     IRKSDGTLQEHDGICEIHVAKYAEIFGLTSAEASKDIRQALKSFAGKEVVFYRPEEDAG
                     DEKGYESFPWFIKRAHSPSRGLYSVHINPYLIPFFIGLQNRFTQFRLSETKEITNPYAM
                     RLYESLCQYRKPDGSGIVSLKIDWIIERYQLPQSYQRMPDFRRRFLQVCVNEINSRTPM
                     RLSYIEKKKGRQTTHIVFSFRDITSMTTG"
     gene            complement(8122..8877)
                     /gene="repE"
                     /label=repE
     repeat_region   8879..9260
                     /gene="ori2"
     gene            8879..9260
                     /gene="ori2"
                     /label=ori2
     rep_origin      8968..9187
                     /label=ori2
                     /note="secondary origin of replication for the bacterial F 
                     plasmid; also known as oriS"
     promoter        9331..9408
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             9400..10491
                     /codon_start=1
                     /gene="lacIq"
                     /product="actose repressor"
                     /label=lacIq
                     /protein_id="BAN79349.1"
                     /translation="MVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAM
                     AELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVER
                     SGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD
                     WSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTED
                     SSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA
                     SPRALADSLMQLARQVSRLESGQ"
     gene            9400..10491
                     /gene="lacIq"
                     /label=lacIq
     CDS             9409..10491
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"