LOCUS       40924_42539        6688 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Fusion protein expression vector pTAG2K, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6688)
  AUTHORS   Keefe AD, Szostak JW.
  TITLE     Functional proteins from a random-sequence library
  JOURNAL   Nature 410 (6829), 715-718 (2001)
  PUBMED    11287961
REFERENCE   2  (bases 1 to 6688)
  AUTHORS   Keefe AD, Wilson DS, Seelig B, Szostak JW.
  TITLE     One-step purification of recombinant proteins using a 
            nanomolar-affinity streptavidin-binding peptide, the SBP-Tag
  JOURNAL   Protein Expr. Purif. 23 (3), 440-446 (2001)
  PUBMED    11722181
REFERENCE   3  (bases 1 to 6688)
  AUTHORS   Keefe AD, Wilson DS, Seelig B, Szostak JW.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-APR-2001) Genetics, Harvard Medical School, 
            Massachusetts General Hospital, Boston, MA 02114, USA
REFERENCE   4  (bases 1 to 6688)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 6688)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature"; 
            date: "2001"; volume: "410"; issue: "6829"; pages: "715-718"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Protein 
            Expr. Purif."; date: "2001"; volume: "23"; issue: "3"; pages: 
            "440-446"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (27-APR-2001) Genetics, Harvard Medical School, Massachusetts 
            General Hospital, Boston, MA 02114, USA"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6688
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             complement(323..340)
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     CDS             complement(347..364)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             complement(374..487)
                     /codon_start=1
                     /label=SBP
                     /note="streptavidin-binding peptide"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             complement(518..1618)
                     /codon_start=1
                     /label=MBP
                     /note="maltose binding protein from E. coli"
                     /translation="MGIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     RBS             complement(1625..1647)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1662..1686)
                     /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)."
     promoter        complement(1687..1705)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2014..2091
                     /label=lacI promoter
     CDS             2092..3171
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    3187..3208
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3983..4171
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    4276..4418
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4604..5192)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             5314..6126
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      complement(6222..6677)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagctttt
      181 agtcgtcatg tccatgatag gtgtcgtccc cgatatcaat gctattgtta aagcaggtct
      241 tacacatgtt atagatcctc aaatgcttgt tcttcacctt ccagttccgg ggagccacct
      301 tgcatttcac acaagggtcc atgctacctc ggggtaccaa gaattcgtga tgatggtgat
      361 ggtgaccgga tcctggttca cgttgacctt gtgggtgatg ctccagtcgt gcacgaagtt
      421 gttcaagttc cccagccagt ccttcaacaa cgtgtccacc tcgccaacca gtggtcttct
      481 cgtccatccc tgagccgcta cctcctgagc tcgaattagt ctgcgcgtct ttcagggctt
      541 catcgacagt ctgacgaccg ctggcggcgt tgatcaccgc agtacgcacg gcataccaga
      601 aagcggacat ctgcgggatg ttcggcatga tttcaccttt ctgggcgttt tccattgtgg
      661 cggcaatacg tggatctttc gccaactctt cctcgtaaga cttcagcgct acggcaccca
      721 gcggtttgtc tttattaacc gcttccagac cttcatcagt cagcagatag ttttcgagga
      781 actcttttgc cagctctttg ttcggactgg cggcgttaat acctgcgctc agcacgccaa
      841 cgaacggttt ggatggttga cccttgaagg tcggcagtac cgttacacca taattcactt
      901 tgctggtgtc gatgttggac catgcccacg ggccgttgat ggtcatcgct gtttcgcctt
      961 tattaaaggc agcttctgcg atggagtaat cggtgtctgc attcatgtgt ttgtttttaa
     1021 tcaggtcaac caggaaggtc agacccgctt tcgcgccagc gttatccacg cccacgtctt
     1081 taatgtcgta cttgccgttt tcatacttga acgcataacc cccgtcagca gcaatcagcg
     1141 gccaggtgaa gtacggttct tgcaggttga acatcagcgc gctcttacct ttcgctttca
     1201 gttctttatc cagcgccggg atctcttccc aggtttttgg cgggttcggc agcagatctt
     1261 tgttataaat cagcgataac gcttcaacag cgatcgggta agcaatcagc ttgccgttgt
     1321 aacgtacggc atcccaggta aacggataca gcttgtcctg gaacgctttg tccggggtga
     1381 tttcagccaa caggccagat tgagcgtagc caccaaagcg gtcgtgtgcc cagaagataa
     1441 tgtcagggcc atcgccagtt gccgcaacct gtgggaattt ctcttccagt ttatccggat
     1501 gctcaacggt gactttaatt ccggtatctt tctcgaattt cttaccgact tcagcgagac
     1561 cgttatagcc tttatcgccg ttaatccaga ttaccagttt accttcttcg attcccatgg
     1621 tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac
     1681 aattccccta tagtgagtcg tattaatttc gcgggatcga gatctcgatc ctctacgccg
     1741 gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg
     1801 acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg
     1861 tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac
     1921 cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc
     1981 aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt
     2041 cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca
     2101 gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg
     2161 gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg
     2221 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
     2281 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
     2341 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
     2401 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
     2461 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
     2521 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
     2581 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
     2641 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
     2701 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
     2761 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
     2821 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
     2881 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
     2941 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
     3001 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
     3061 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     3121 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     3181 cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag
     3241 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     3301 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat
     3361 tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt
     3421 cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg
     3481 cgagaagcag gccattatcg ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct
     3541 gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc
     3601 gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac
     3661 atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg
     3721 caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac
     3781 atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat
     3841 ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt
     3901 aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa
     3961 tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg
     4021 ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga
     4081 acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct
     4141 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     4201 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     4261 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     4321 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatatg cggtgtgaaa
     4381 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     4441 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4501 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4561 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4621 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4681 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4741 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4801 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4861 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4921 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4981 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5041 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5101 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5161 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5221 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg
     5281 tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct
     5341 tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     5401 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg
     5461 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     5521 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     5581 actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta
     5641 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     5701 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     5761 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     5821 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     5881 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     5941 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     6001 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     6061 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     6121 tttttctaag aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6181 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaaattgtaa acgttaatat
     6241 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     6301 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     6361 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     6421 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     6481 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     6541 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     6601 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     6661 gccgctacag ggcgcgtccc attcgcca
//
