LOCUS       40924_18461        7316 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pET28_nusA_tjp1aN, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7316)
  AUTHORS   Fadeev A, Irion U, Nuesslein-Volhard C, Krauss J, Frohnhoefer HG.
  TITLE     Direct Submission
  JOURNAL   Submitted (06-APR-2015) Arbeitsgruppe Nuesslein-Volhard, Max Planck 
            Institute for Developmental Biology, Spemannstrasse 35, Tuebingen, 
            Germany 72076, Germany
REFERENCE   2  (bases 1 to 7316)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7316)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Submitted 
            (06-APR-2015) Arbeitsgruppe Nuesslein-Volhard, Max Planck Institute 
            for Developmental Biology, Spemannstrasse 35, Tuebingen, Germany 
            72076, Germany"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7316
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             13..30
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             40..57
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     CDS             67..1545
                     /codon_start=1
                     /label=NusA
                     /note="transcription elongation protein (N utilization
                     substance protein A)"
                     /translation="KEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDVRV
                     QIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDRIT
                     TQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVILRE
                     DMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIEIK
                     AAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPAQF
                     VINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMTVD
                     DLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLDEP
                     TVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLEDLA
                     EQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA"
     CDS             1561..1578
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     misc_feature    1585..2040
                     /label=Region: tjp1a
                     /note="Region: tjp1a"
     CDS             2090..2107
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      2174..2221
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2258..2713
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(2809..3621)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      3743..4331
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4517..4659)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4764..4952)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5727..5748)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5764..6843)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6844..6921)
                     /label=lacI promoter
     promoter        7230..7248
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7249..7273
                     /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             7288..7310
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat
       61 aatatgaaag aaattttggc tgtagttgaa gccgtatcca atgaaaaggc gctacctcgc
      121 gagaagattt tcgaagcatt ggaaagcgcg ctggcgacag caacaaagaa aaaatatgaa
      181 caagagatcg acgtccgcgt acagatcgat cgcaaaagcg gtgattttga cactttccgt
      241 cgctggttag ttgttgatga agtcacccag ccgaccaagg aaatcaccct tgaagccgca
      301 cgttatgaag atgaaagcct gaacctgggc gattacgttg aagatcagat tgagtctgtt
      361 acctttgacc gtatcactac ccagacggca aaacaggtta tcgtgcagaa agtgcgtgaa
      421 gccgaacgtg cgatggtggt tgatcagttc cgtgaacacg aaggtgaaat catcaccggc
      481 gtggtgaaaa aagtaaaccg cgacaacatc tctctggatc tgggcaacaa cgctgaagcc
      541 gtgatcctgc gcgaagatat gctgccgcgt gaaaacttcc gccctggcga ccgcgttcgt
      601 ggcgtgctct attccgttcg cccggaagcg cgtggcgcgc aactgttcgt cactcgttcc
      661 aagccggaaa tgctgatcga actgttccgt attgaagtgc cagaaatcgg cgaagaagtg
      721 attgaaatta aagcagcggc tcgcgatccg ggttctcgtg cgaaaatcgc ggtgaaaacc
      781 aacgataaac gtatcgatcc ggtaggtgct tgcgtaggta tgcgtggcgc gcgtgttcag
      841 gcggtgtcta ctgaactggg tggcgagcgt atcgatatcg tcctgtggga tgataacccg
      901 gcgcagttcg tgattaacgc aatggcaccg gcagacgttg cttctatcgt ggtggatgaa
      961 gataaacaca ctatggacat cgccgttgaa gccggtaatc tggcgcaggc gattggccgt
     1021 aacggtcaga acgtgcgtct ggcttcgcaa ctgagcggtt gggaactcaa cgtgatgacc
     1081 gttgacgacc tgcaagctaa gcatcaggcg gaagcgcacg cagcgatcga caccttcacc
     1141 aaatatctcg acatcgacga agacttcgcg actgttctgg tagaagaagg cttctcgacg
     1201 ctggaagaat tggcctatgt gccgatgaaa gagctgttgg aaatcgaagg ccttgatgag
     1261 ccgaccgttg aagcactgcg cgagcgtgct aaaaatgcac tggccaccat tgcacaggcc
     1321 caggaagaaa gcctcggtga taacaaaccg gctgacgatc tgctgaacct tgaaggggta
     1381 gatcgtgatt tggcattcaa actggccgcc cgtggcgttt gtacgctgga agatctcgcc
     1441 gaacagggca ttgatgatct ggctgatatc gaagggttga ccgacgaaaa agccggagca
     1501 ctgattatgg ctgcccgtaa tatttgctgg ttcggtgacg aagcgactag tggttctggt
     1561 ctggtgccgc gcggcagcca tatgtacaag aaggatatct accgacccaa cgaaccggtg
     1621 cagataagcc aaaaccctat gcagcccaac tactaccagg aagacaagcc atacagagat
     1681 tatgaccacc agccctaccg ctatgatgga ggctacatgg aaccaaaggc tcgtaacttt
     1741 gacccacatt ttgaggacag cgtgcctcca tataatgatc aatggggtca gtatgaccac
     1801 acctctgggt atgaacctca aacgctctac gaggatggtc tgaaccgctt gtatggtcac
     1861 cctcagtcac gaaacgacac cggctacgac acaggtcgac ctcgatatgg caaaccaacc
     1921 tctggaccaa tccgacacga tgaaccacca ccatcacgcc cacaatatga cactcaacaa
     1981 acctctcgtt ccccagaacc gcccaaacaa caatactacg acccttccca aaggccttcc
     2041 taaggatccg aattcgagct ccgtcgacaa gcttgcggcc gcactcgagc accaccacca
     2101 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     2161 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     2221 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     2281 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2341 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2401 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2461 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2521 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2581 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2641 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     2701 acgtttacaa tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     2761 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     2821 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     2881 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     2941 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     3001 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     3061 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     3121 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     3181 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     3241 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     3301 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     3361 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     3421 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     3481 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     3541 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     3601 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     3661 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3721 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3781 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3841 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3901 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3961 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4021 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4081 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4141 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4201 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4261 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4321 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4381 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4441 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4501 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4561 accgcatata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     4621 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     4681 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4741 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     4801 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     4861 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     4921 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     4981 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     5041 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     5101 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     5161 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     5221 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     5281 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     5341 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     5401 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     5461 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     5521 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     5581 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     5641 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     5701 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     5761 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5821 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     5881 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     5941 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     6001 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     6061 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     6121 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     6181 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     6241 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     6301 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     6361 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     6421 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     6481 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     6541 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     6601 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     6661 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     6721 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     6781 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     6841 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     6901 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     6961 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     7021 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     7081 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     7141 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     7201 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     7261 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacc
//
