LOCUS       40924_33177        8593 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNGWA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8593)
  AUTHORS   Busso D, Delagoutte-Busso B, Moras D.
  TITLE     Construction of a set Gateway-based destination vectors for 
            high-throughput cloning and expression screening in Escherichia coli
  JOURNAL   Anal. Biochem. 343 (2), 313-321 (2005)
  PUBMED    15993367
REFERENCE   2  (bases 1 to 8593)
  AUTHORS   Busso D, Delagoutte-Busso B, Salim L, Moras D.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-APR-2008) Structural Biology and Genomics Platform, 
            IGBMC, 1, rue Laurent Fries, Illkirch 67404, France
REFERENCE   3  (bases 1 to 8593)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8593)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Anal. 
            Biochem."; date: "2005"; volume: "343"; issue: "2"; pages: "313-321"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (25-APR-2008) Structural Biology and Genomics Platform, IGBMC, 1, 
            rue Laurent Fries, Illkirch 67404, France"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        27..45
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    46..70
                     /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             85..107
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             123..1604
                     /codon_start=1
                     /label=NusA
                     /note="transcription elongation protein (N utilization
                     substance protein A)"
                     /translation="NKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDVR
                     VQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDRI
                     TTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVILR
                     EDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIEI
                     KAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPAQ
                     FVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMTV
                     DDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLDE
                     PTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLEDL
                     AEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA"
     protein_bind    1623..1747
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1772..1802
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1856..2512
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2857..3159
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3203..3327)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3341..3358
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      3425..3472
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3509..3964
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3991..4095
                     /label=AmpR promoter
     CDS             4096..4953
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5127..5715
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(5901..6043)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(6148..6336)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(7111..7132)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(7148..8227)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(8228..8305)
                     /label=lacI promoter
ORIGIN
        1 agatccagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
       61 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgggta
      121 ccaacaaaga aattttggct gtagttgaag ccgtatccaa tgaaaaggcg ctacctcgcg
      181 agaagatttt cgaagcattg gaaagcgcgc tggcgacagc aacaaagaaa aaatatgaac
      241 aagagatcga cgtccgcgta cagatcgatc gcaaaagcgg tgattttgac actttccgtc
      301 gctggttagt tgttgatgaa gtcacccagc cgaccaagga aatcaccctt gaagccgcac
      361 gttatgaaga tgaaagcctg aacctgggcg attacgttga agatcagatt gagtctgtta
      421 cctttgaccg tatcactacc cagacggcaa aacaggttat cgtgcagaaa gtgcgtgaag
      481 ccgaacgtgc gatggtggtt gatcagttcc gtgaacacga aggtgaaatc atcaccggcg
      541 tggtgaaaaa agtaaaccgc gacaacatct ctctggatct gggcaacaac gctgaagccg
      601 tgatcctgcg cgaagatatg ctgccgcgtg aaaacttccg ccctggcgac cgcgttcgtg
      661 gcgtgctcta ttccgttcgc ccggaagcgc gtggcgcgca actgttcgtc actcgttcca
      721 agccggaaat gctgatcgaa ctgttccgta ttgaagtgcc agaaatcggc gaagaagtga
      781 ttgaaattaa agcagcggct cgcgatccgg gttctcgtgc gaaaatcgcg gtgaaaacca
      841 acgataaacg tatcgatccg gtaggtgctt gcgtaggtat gcgtggcgcg cgtgttcagg
      901 cggtgtctac tgaactgggt ggcgagcgta tcgatatcgt cctgtgggat gataacccgg
      961 cgcagttcgt gattaacgca atggcaccgg cagacgttgc ttctatcgtg gtggatgaag
     1021 ataaacacac catggacatc gccgttgaag ccggtaatct ggcgcaggcg attggccgta
     1081 acggtcagaa cgtgcgtctg gcttcgcaac tgagcggttg ggaactcaac gtgatgaccg
     1141 ttgacgacct gcaagctaag catcaggcgg aagcgcacgc agcgatcgac accttcacca
     1201 aatatctcga catcgacgaa gacttcgcga ctgttctggt agaagaaggc ttctcgacgc
     1261 tggaagaatt ggcctatgtg ccgatgaaag agctgttgga aatcgaaggc cttgatgagc
     1321 cgaccgttga agcactgcgc gagcgtgcta aaaatgcact ggccaccatt gcacaggccc
     1381 aggaagaaag cctcggtgat aacaaaccgg ctgacgatct gctgaacctt gaaggggtag
     1441 atcgtgattt ggcattcaaa ctggccgccc gtggcgtttg tacgctggaa gatctcgccg
     1501 aacagggcat tgatgatctg gctgatatcg aagggttgac cgacgaaaaa gccggagcac
     1561 tgattatggc tgcccgtaat atttgctggt tcggtgacga agcgggtacc ggatcttaca
     1621 tcacaagttt gtacaaaaaa gctgaacgag aaacgtaaaa tgatataaat atcaatatat
     1681 taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt
     1741 cactatggcg gccgcattag gcaccccagg ctttacactt tatgcttccg gctcgtataa
     1801 tgtgtggatt ttgagttagg atccgtcgag attttcagga gctaaggaag ctaaaatgga
     1861 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt
     1921 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac
     1981 ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat
     2041 tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag acggtgagct
     2101 ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt
     2161 ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca
     2221 agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat
     2281 gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa
     2341 tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa
     2401 ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg
     2461 cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaaacgcg
     2521 tggatccggc ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg atttttgcgg
     2581 tataagaata tatactgata tgtatacccg aagtatgtca aaaagaggta tgctatgaag
     2641 cagcgtatta cagtgacagt tgacagcgac agctatcagt tgctcaaggc atatatgatg
     2701 tcaatatctc cggtctggta agcacaacca tgcagaatga agcccgtcgt ctgcgtgccg
     2761 aacgctggaa agcggaaaat caggaaggga tggctgaggt cgcccggttt attgaaatga
     2821 acggctcttt tgctgacgag aacaggggct ggtgaaatgc agtttaaggt ttacacctat
     2881 aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccc
     2941 gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt
     3001 gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg
     3061 gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat
     3121 gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg ctcccttata
     3181 cacagccagt ctgcaggtcg accatagtga ctggatatgt tgtgttttac agtattatgt
     3241 agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc
     3301 tcgttcagct ttcttgtaca aagtggtgat gtacctcgag caccaccacc accaccactg
     3361 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     3421 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     3481 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     3541 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3601 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3661 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3721 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3781 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3841 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     3901 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     3961 atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4021 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4081 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4141 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4201 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     4261 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     4321 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     4381 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     4441 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     4501 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     4561 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     4621 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     4681 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     4741 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     4801 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     4861 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     4921 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4981 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5041 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5101 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5161 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5221 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     5281 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5341 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5401 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5461 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5521 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5581 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5641 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5701 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5761 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     5821 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     5881 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5941 tcacaccgca tatatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     6001 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     6061 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     6121 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     6181 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     6241 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     6301 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     6361 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     6421 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     6481 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     6541 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     6601 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     6661 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     6721 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     6781 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     6841 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     6901 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     6961 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     7021 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     7081 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     7141 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     7201 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     7261 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     7321 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     7381 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     7441 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     7501 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     7561 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     7621 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     7681 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     7741 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     7801 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     7861 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     7921 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     7981 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     8041 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     8101 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     8161 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     8221 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     8281 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     8341 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     8401 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     8461 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     8521 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     8581 gcgtagagga tcg
//
