LOCUS       40924_18191        7440 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Gateway(R) destination vector for high-level T7-regulated bacterial 
            expression and C-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7440)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7440)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        318..336
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    337..361
                     /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)."
     protein_bind    402..526
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        551..581
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             635..1291
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             1636..1938
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(1982..2106)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2126..2167
                     /codon_start=1
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
                     /translation="GKPIPNPLLGLDST"
     CDS             2177..2194
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      2265..2312
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(2487..2515)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     promoter        2632..2736
                     /label=AmpR promoter
     CDS             2737..3594
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3768..4356
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4542..4684)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4789..4977)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(6255..6276)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(6292..7371)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(join(7372..7440,1..9))
                     /label=lacI promoter
ORIGIN
        1 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
       61 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
      121 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
      181 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
      241 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
      301 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
      361 ccctctagaa ataattttgt ttaactttaa gaaggaatat cacaagtttg tacaaaaaag
      421 ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa
      481 aacagactac ataatactgt aaaacacaac atatccagtc actatggcgg ccgcattagg
      541 caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggattt tgagttagga
      601 tccggcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
      661 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc
      721 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa
      781 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
      841 tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca
      901 cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata
      961 ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga
     1021 aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc
     1081 ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc
     1141 cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat
     1201 tcaggttcat catgccgtct gtgatggctt ccatgtcggc agaatgctta atgaattaca
     1261 acagtactgc gatgagtggc agggcggggc gtaaacgcgt ggatccggct tactaaaagc
     1321 cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat
     1381 gtatacccga agtatgtcaa aaagaggtgt gctatgaagc agcgtattac agtgacagtt
     1441 gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa
     1501 gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc
     1561 aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga
     1621 acagggactg gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt
     1681 ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc
     1741 ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat
     1801 atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt
     1861 atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac
     1921 ctgatgttct ggggaatata aatgtcaggc tccgttatac acagccagtc tgcaggtcga
     1981 ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat
     2041 ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt tcttgtacaa
     2101 agtggtgatc aattcgaagc ttgaaggtaa gcctatccct aaccctctcc tcggtctcga
     2161 ttctacgcgt accggtcatc atcaccatca ccattgagtt tgatccggct gctaacaaag
     2221 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     2281 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc
     2341 ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac
     2401 ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta
     2461 gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg
     2521 agaattaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     2581 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     2641 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     2701 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     2761 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     2821 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     2881 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     2941 acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa
     3001 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3061 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3121 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3181 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3241 gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg
     3301 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3361 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3421 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     3481 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     3541 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     3601 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     3661 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     3721 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3781 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3841 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3901 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3961 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4021 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4081 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4141 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4201 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4261 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4321 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4381 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4441 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4501 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4561 cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt acaatctgct
     4621 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     4681 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4741 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4801 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     4861 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     4921 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     4981 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     5041 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     5101 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     5161 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     5221 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     5281 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     5341 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     5401 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct
     5461 gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca
     5521 agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt
     5581 ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat
     5641 gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc
     5701 aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccagtg
     5761 atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca
     5821 tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga
     5881 agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc
     5941 agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     6001 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     6061 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     6121 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     6181 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     6241 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     6301 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     6361 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     6421 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     6481 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     6541 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     6601 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     6661 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     6721 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     6781 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     6841 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     6901 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6961 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     7021 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     7081 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     7141 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     7201 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     7261 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     7321 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     7381 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
//
