LOCUS       40924_24767        8620 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pHNGWA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8620)
  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 8620)
  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 8620)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8620)
  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..8620
                     /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             126..143
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             150..1631
                     /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    1650..1774
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1799..1829
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1883..2539
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2884..3186
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3230..3354)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3368..3385
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      3452..3499
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3536..3991
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        4018..4122
                     /label=AmpR promoter
     CDS             4123..4980
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5154..5742
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(5928..6070)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(6175..6363)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(7138..7159)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(7175..8254)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(8255..8332)
                     /label=lacI promoter
ORIGIN
        1 agatccagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
       61 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgggca
      121 gcagccatca tcatcatcat cacggtacca acaaagaaat tttggctgta gttgaagccg
      181 tatccaatga aaaggcgcta cctcgcgaga agattttcga agcattggaa agcgcgctgg
      241 cgacagcaac aaagaaaaaa tatgaacaag agatcgacgt ccgcgtacag atcgatcgca
      301 aaagcggtga ttttgacact ttccgtcgct ggttagttgt tgatgaagtc acccagccga
      361 ccaaggaaat cacccttgaa gccgcacgtt atgaagatga aagcctgaac ctgggcgatt
      421 acgttgaaga tcagattgag tctgttacct ttgaccgtat cactacccag acggcaaaac
      481 aggttatcgt gcagaaagtg cgtgaagccg aacgtgcgat ggtggttgat cagttccgtg
      541 aacacgaagg tgaaatcatc accggcgtgg tgaaaaaagt aaaccgcgac aacatctctc
      601 tggatctggg caacaacgct gaagccgtga tcctgcgcga agatatgctg ccgcgtgaaa
      661 acttccgccc tggcgaccgc gttcgtggcg tgctctattc cgttcgcccg gaagcgcgtg
      721 gcgcgcaact gttcgtcact cgttccaagc cggaaatgct gatcgaactg ttccgtattg
      781 aagtgccaga aatcggcgaa gaagtgattg aaattaaagc agcggctcgc gatccgggtt
      841 ctcgtgcgaa aatcgcggtg aaaaccaacg ataaacgtat cgatccggta ggtgcttgcg
      901 taggtatgcg tggcgcgcgt gttcaggcgg tgtctactga actgggtggc gagcgtatcg
      961 atatcgtcct gtgggatgat aacccggcgc agttcgtgat taacgcaatg gcaccggcag
     1021 acgttgcttc tatcgtggtg gatgaagata aacacaccat ggacatcgcc gttgaagccg
     1081 gtaatctggc gcaggcgatt ggccgtaacg gtcagaacgt gcgtctggct tcgcaactga
     1141 gcggttggga actcaacgtg atgaccgttg acgacctgca agctaagcat caggcggaag
     1201 cgcacgcagc gatcgacacc ttcaccaaat atctcgacat cgacgaagac ttcgcgactg
     1261 ttctggtaga agaaggcttc tcgacgctgg aagaattggc ctatgtgccg atgaaagagc
     1321 tgttggaaat cgaaggcctt gatgagccga ccgttgaagc actgcgcgag cgtgctaaaa
     1381 atgcactggc caccattgca caggcccagg aagaaagcct cggtgataac aaaccggctg
     1441 acgatctgct gaaccttgaa ggggtagatc gtgatttggc attcaaactg gccgcccgtg
     1501 gcgtttgtac gctggaagat ctcgccgaac agggcattga tgatctggct gatatcgaag
     1561 ggttgaccga cgaaaaagcc ggagcactga ttatggctgc ccgtaatatt tgctggttcg
     1621 gtgacgaagc gggtaccgga tcttacatca caagtttgta caaaaaagct gaacgagaaa
     1681 cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa cagactacat
     1741 aatactgtaa aacacaacat atccagtcac tatggcggcc gcattaggca ccccaggctt
     1801 tacactttat gcttccggct cgtataatgt gtggattttg agttaggatc cgtcgagatt
     1861 ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat
     1921 atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta
     1981 taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca
     2041 caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt
     2101 ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac
     2161 cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt
     2221 ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta
     2281 tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt
     2341 caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat
     2401 gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca
     2461 tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga
     2521 tgagtggcag ggcggggcgt aaacgcgtgg atccggctta ctaaaagcca gataacagta
     2581 tgcgtatttg cgcgctgatt tttgcggtat aagaatatat actgatatgt atacccgaag
     2641 tatgtcaaaa agaggtatgc tatgaagcag cgtattacag tgacagttga cagcgacagc
     2701 tatcagttgc tcaaggcata tatgatgtca atatctccgg tctggtaagc acaaccatgc
     2761 agaatgaagc ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag gaagggatgg
     2821 ctgaggtcgc ccggtttatt gaaatgaacg gctcttttgc tgacgagaac aggggctggt
     2881 gaaatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat
     2941 gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct ggccagtgca
     3001 cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa
     3061 agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa
     3121 gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg
     3181 ggaatataaa tgtcaggctc ccttatacac agccagtctg caggtcgacc atagtgactg
     3241 gatatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct aatttaatat
     3301 attgatattt atatcatttt acgtttctcg ttcagctttc ttgtacaaag tggtgatgta
     3361 cctcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     3421 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     3481 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     3541 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3601 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3661 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3721 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3781 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3841 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3901 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3961 aattttaaca aaatattaac gtttacaatt tcaggtggca cttttcgggg aaatgtgcgc
     4021 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4081 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4141 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4201 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4261 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4321 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4381 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4441 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4501 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4561 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4621 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     4681 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4741 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4801 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     4861 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4921 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4981 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     5041 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5101 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5161 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5221 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5281 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     5341 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5401 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5461 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5521 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5581 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5641 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5701 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5761 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     5821 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     5881 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     5941 tttctcctta cgcatctgtg cggtatttca caccgcatat atggtgcact ctcagtacaa
     6001 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     6061 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     6121 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     6181 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     6241 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     6301 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     6361 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     6421 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     6481 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     6541 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     6601 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     6661 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     6721 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     6781 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     6841 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     6901 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     6961 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     7021 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     7081 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     7141 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     7201 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     7261 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     7321 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     7381 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     7441 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     7501 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     7561 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     7621 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     7681 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     7741 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     7801 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     7861 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     7921 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     7981 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     8041 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     8101 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     8161 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     8221 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     8281 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     8341 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     8401 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     8461 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     8521 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     8581 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
//
