LOCUS       40924_18296        7650 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Gateway(R) destination vector for high-level inducible expression of
            biotinylated proteins in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7650)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7650)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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)."
     RBS             267..289
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             297..329
                     /codon_start=1
                     /label=T7 tag (gene 10 leader)
                     /note="leader peptide from bacteriophage T7 gene 10"
                     /translation="MASMTGGQQMG"
     CDS             336..551
                     /codon_start=1
                     /label=BioEase(TM) tag
                     /note="biotinylated sequence derived from the C-terminus of
                     the alpha-subunit of Klebsiella pneumoniae oxaloacetate 
                     decarboxylase"
                     /translation="GAGTPVTAPLAGTIWKVLASEGQTVAAGEVLLILEAMKMETEIRA
                     AQAGTVRGIAVKAGDAVAVGDTLMTLA"
     CDS             564..587
                     /codon_start=1
                     /label=Xpress(TM) tag
                     /note="Xpress(TM) epitope tag, including an enterokinase 
                     recognition and cleavage site"
                     /translation="DLYDDDDK"
     protein_bind    597..721
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        746..776
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             830..1486
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             1810..2112
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(2156..2280)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      2362..2409
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        2733..2837
                     /label=AmpR promoter
     CDS             2838..3695
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3869..4457
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4643..4785)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4890..5078)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(6356..6377)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(6393..7472)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7473..7550)
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgg
      301 ctagcatgac tggtggacag caaatgggta ttatgggcgc cggcaccccg gtgaccgccc
      361 cgctggcggg cactatctgg aaggtgctgg ccagcgaagg ccagacggtg gccgcaggcg
      421 aggtgctgct gattctggaa gccatgaaga tggaaaccga aatccgcgcc gcgcaggccg
      481 ggaccgtgcg cggtatcgcg gtgaaagccg gcgacgcggt ggcggtcggc gacaccctga
      541 tgaccctggc gggctctgga tccgatctgt acgacgatga cgataaggga attatcacaa
      601 gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat atattaaatt
      661 agattttgca taaaaaacag actacataat actgtaaaac acaacatatc cagtcactat
      721 ggcggccgca ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg
      781 gattttgagt taggatccgt cgagattttc aggagctaag gaagctaaaa tggagaaaaa
      841 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
      901 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt
      961 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
     1021 ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat
     1081 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc
     1141 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt
     1201 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt
     1261 cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga
     1321 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct
     1381 gatgccgctg gcgattcagg ttcatcatgc cgtctgtgat ggcttccatg tcggcagaat
     1441 gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaaa cgcgtggatc
     1501 cggcttacta aaagccagat aacagtatgc gtatttgcgc gcaccggtgc tagcgtatac
     1561 ccgaagtatg tcaaaaagag gtgtgctatg aagcagcgta ttacagtgac agttgacagc
     1621 gacagctatc agttgctcaa ggcatatatg atgtcaatat ctccggtctg gtaagcacaa
     1681 ccatgcagaa tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag
     1741 ggatggctga ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac gagaacaggg
     1801 actggtgaaa tgcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt
     1861 gtggatgtac agagtgatat tattgacacg cccgggcgac ggatggtgat ccccctggcc
     1921 agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg
     1981 gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg
     2041 gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg
     2101 ttctggggaa tataaatgtc aggctccgtt atacacagcc agtctgcagg tcgaccatag
     2161 tgactggata tgttgtgttt tacagtatta tgtagtctgt tttttatgca aaatctaatt
     2221 taatatattg atatttatat cattttacgt ttctcgttca gctttcttgt acaaagtggt
     2281 gataattaat taagatcaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     2341 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     2401 ggttttttgc tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg
     2461 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
     2521 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
     2581 cgcattaaag ctagcttatc gatgataagc tgtcaaacat gagaattaat tcttgaagac
     2641 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     2701 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2761 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2821 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     2881 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     2941 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3001 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3061 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     3121 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3181 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3241 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3301 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3361 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3421 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3481 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3541 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3601 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3661 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3721 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3781 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3841 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3901 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3961 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     4021 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4081 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4141 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4201 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4261 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4321 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4381 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4441 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4501 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4561 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4621 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4681 tttcacaccg catatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4741 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4801 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4861 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4921 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     4981 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     5041 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     5101 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     5161 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     5221 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     5281 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     5341 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     5401 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     5461 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     5521 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
     5581 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
     5641 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
     5701 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
     5761 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
     5821 ccgaggcggc ataaatcgcc gtgacgatca gcggtccagt gatcgaagtt aggctggtaa
     5881 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
     5941 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
     6001 ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc
     6061 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     6121 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     6181 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     6241 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     6301 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     6361 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6421 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6481 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     6541 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     6601 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     6661 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     6721 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     6781 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     6841 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     6901 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     6961 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     7021 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     7081 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     7141 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     7201 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     7261 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     7321 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     7381 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     7441 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     7501 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     7561 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     7621 gagcaccgcc gccgcaagga atggtgcatg
//
