LOCUS       Exported               10726 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Helper vector pTKRED, complete sequence.
ACCESSION   GU327533
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10726)
  AUTHORS   Kuhlman TE, Cox EC.
  TITLE     Site-specific chromosomal integration of large synthetic constructs
  JOURNAL   Nucleic Acids Res. 38 (6), E92 (2010)
  PUBMED    20047970
REFERENCE   2  (bases 1 to 10726)
  AUTHORS   Kuhlman TE, Cox EC.
  TITLE     Direct Submission
  JOURNAL   Submitted (17-DEC-2009) Molecular Biology, Princeton University, 
            Washington Rd., Princeton, NJ 08544, USA
REFERENCE   3  (bases 1 to 10726)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10726
                     /organism="Helper vector pTKRED"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /db_xref="taxon:711475"
     CDS             complement(144..1022)
                     /codon_start=1
                     /gene="araC"
                     /product="AraC"
                     /label=araC
                     /protein_id="ADB98007.1"
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     gene            complement(144..1022)
                     /gene="araC"
                     /label=araC
     promoter        1049..1333
                     /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)"
     regulatory      complement(1173..1201)
                     /regulatory_class="promoter"
                     /note="ParaC"
     regulatory      1298..1325
                     /regulatory_class="promoter"
                     /note="ParaBAD"
     RBS             1360..1368
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3 end of the 16S rRNA (Chen et al., 1994)"
     CDS             1377..2078
                     /codon_start=1
                     /gene="I-SceI"
                     /product="I-SceI"
                     /label=I-SceI
                     /protein_id="ADB98008.1"
                     /translation="MHQKNQVMNLGPNSKLLKEYKSQLIELNIEQFEAGIGLILGDAYI
                     RSRDEGKTYCMQFEWKNKAYMDHVCLLYDQWVLSPPHKKQRVNHLGNLVITWGAQTFKH
                     QAFNKLANLFIVNNKKTIPNNLVENYLTPMSLAYWFMDDGGKWDYNKNSTNKSIVLNTQ
                     SFTFEEVEYLVKGLRNKFQLNCYVKINKNKPIIYIDSMSYLIFYNLIKPYLIPQMMYKL
                     PNTISSETFLK"
     gene            1377..2078
                     /gene="I-SceI"
                     /label=I-SceI
     CDS             2444..3454
                     /codon_start=1
                     /gene="aadA"
                     /product="AadA"
                     /function="spectinomycin resistance"
                     /label=aadA
                     /protein_id="ADB98009.1"
                     /translation="MRSRNWSRTLTERSGGNGAVAVFMACYDCFFGVQSMPRASKQQAR
                     YAVGRCLMLWSSNDVTQQGSRPKTKLNIMREAVIAEVSTQLSEVVGVIERHLEPTLLAV
                     HLYGSAVDGGLKPHSDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTI
                     VVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPA
                     AEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVA
                     ADWAMERLPAQYQPVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     gene            2444..3454
                     /gene="aadA"
                     /label=aadA
     CDS             2663..3454
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     CDS             complement(3778..4458)
                     /codon_start=1
                     /gene="exo"
                     /product="Exo"
                     /label=exo
                     /protein_id="ADB98010.1"
                     /translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     gene            complement(3778..4458)
                     /gene="exo"
                     /label=exo
     CDS             complement(4455..5240)
                     /codon_start=1
                     /gene="beta"
                     /product="Beta"
                     /label=beta
                     /protein_id="ADB98011.1"
                     /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
                     ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
                     TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
                     FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
                     LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
     gene            complement(4455..5240)
                     /gene="beta"
                     /label=beta
     CDS             complement(5246..5662)
                     /codon_start=1
                     /gene="gam"
                     /product="Gam"
                     /label=gam
                     /protein_id="ADB98012.1"
                     /translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
                     IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
                     AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
     gene            complement(5246..5662)
                     /gene="gam"
                     /label=gam
     regulatory      complement(5687..5714)
                     /regulatory_class="promoter"
                     /note="Plac"
     protein_bind    5696..5712
                     /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(5720..5750)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             complement(5876..6937)
                     /codon_start=1
                     /gene="recA"
                     /product="RecA"
                     /label=recA
                     /protein_id="ADB98013.1"
                     /translation="MAIDENKQKALAAALGQIEKQFGKGSIMRLGEDRSMDVETISTGS
                     LSLDIALGAGGLPMGRIVEIYGPESSGKTTLTLQVIAAAQREGKTCAFIDAEHALDPIY
                     ARKLGVDIDNLLCSQPDTGEQALEICDALARSGAVDVIVVDSVAALTPKAEIEGEIGDS
                     HMGLAARMMSQAMRKLAGNLKQSNTLLIFINQIRMKIGVMFGNPETTTGGNALKFYASV
                     RLDIRRIGAVKEGENVVGSETRVKVVKNKIAAPFKQAEFQILYGEGINFYGELVDLGVK
                     EKLIEKAGAWYSYKGEKIGQGKANATAWLKDNPETAKEIEKKVRELLLSNPNSTPDFSV
                     DDSEGVAETNEDF"
     gene            complement(5876..6937)
                     /gene="recA"
                     /label=recA
     CDS             complement(7426..8508)
                     /codon_start=1
                     /gene="lacI"
                     /product="LacI"
                     /label=lacI
                     /protein_id="ADB98014.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(7426..8508)
                     /gene="lacI"
                     /label=lacI
     regulatory      complement(8542..8574)
                     /regulatory_class="promoter"
                     /note="PlacI"
     CDS             complement(8694..9644)
                     /codon_start=1
                     /gene="repA101"
                     /product="RepA101"
                     /label=repA101
                     /protein_id="ADB98015.1"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     gene            complement(8694..9644)
                     /gene="repA101"
                     /label=repA101
     rep_origin      9648..10388
                     /label=pSC101 ori
                     /note="pSC101 ori"
     rep_origin      9692..9914
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"