LOCUS       40924_18456        7172 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pET28_nusA_tjp1aC, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7172)
  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 7172)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7172)
  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..7172
                     /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..1896
                     /label=Region: tjp1a
                     /note="Region: tjp1a"
     CDS             1946..1963
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      2030..2077
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2114..2569
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(2665..3477)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      3599..4187
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4373..4515)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4620..4808)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5583..5604)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5620..6699)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6700..6777)
                     /label=lacI promoter
     promoter        7086..7104
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7105..7129
                     /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             7144..7166
                     /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 tatgaaaccc tccacacagc tgacacctgc catcaaacca
     1621 gcccaacccc aaactcaacc agcacactat cccagagcag agtcagtgga gaaaatcaac
     1681 ccagctgctc catcaaaccc agctgtccca ccaccaaccc tgcctaaacc tgcagcaacc
     1741 ccacctctcg accctcacgg atctcccaaa gtaaagcctc ctagtcatga agacacagtc
     1801 cagagcactt acctgcctca caaaagcttc ccagagaaat cccccgtcaa cggcaccggc
     1861 gaacctccaa agaccaacag cactgccacg tccagctaag gatccgaatt cgagctccgt
     1921 cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     1981 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     2041 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     2101 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2161 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2221 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2281 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2341 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2401 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2461 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2521 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc aggtggcact
     2581 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2641 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     2701 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     2761 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     2821 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     2881 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     2941 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     3001 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     3061 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     3121 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     3181 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     3241 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     3301 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     3361 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     3421 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     3481 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     3541 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3601 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3661 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3721 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3781 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3841 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3901 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3961 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4021 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4081 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4141 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4201 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4261 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4321 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     4381 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatatggt gcactctcag
     4441 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     4501 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4561 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4621 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     4681 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     4741 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     4801 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     4861 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     4921 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     4981 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     5041 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     5101 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     5161 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     5221 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     5281 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     5341 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     5401 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     5461 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     5521 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     5581 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5641 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5701 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     5761 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     5821 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     5881 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     5941 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     6001 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     6061 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     6121 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     6181 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     6241 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     6301 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     6361 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     6421 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     6481 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     6541 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     6601 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     6661 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     6721 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     6781 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     6841 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     6901 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     6961 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     7021 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     7081 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     7141 aattttgttt aactttaaga aggagatata cc
//
