LOCUS       40924_18636        5612 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Flexi(R) Vector with the barnase gene, for in vitro transcription 
            and translation of a protein with an N-terminal GST-HRV 3C cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5612)
  AUTHORS   Vinarov DA, Lytle BL, Peterson FC, Tyler EM, Volkman BF, Markley JL.
  TITLE     Cell-free protein production and labeling protocol for NMR-based 
            structural proteomics.
  JOURNAL   Nat. Methods 2004;1:149-53.
  PUBMED    15782178
REFERENCE   2  (bases 1 to 5612)
  AUTHORS   Center for Eukaryotic Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5612)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Methods"; date: "2004"; volume: "1"; pages: "149-53"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     To remove the lethal barnase gene, insert an ORF between the SgfI 
            site and the PmeI site or a downstream site.
FEATURES             Location/Qualifiers
     source          1..5612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     CDS             155..805
                     /codon_start=1
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             812..835
                     /codon_start=1
                     /label=HRV 3C site
                     /note="recognition and cleavage site for human rhinovirus
                     3C and PreScission proteases"
                     /translation="LEVLFQGP"
     CDS             867..1199
                     /codon_start=1
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
                     /translation="MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLAD
                     VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHY
                     QTFTKIR"
     promoter        1218..1248
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1302..1958
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     misc_feature    1971..2026
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2697..2713)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2721..2737)
                     /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(2745..2775)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2790..2811)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3099..3687)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3861..4718)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4719..4823)
                     /label=AmpR promoter
ORIGIN
        1 atttaggtga cactatagaa tacacggaat tcgcgttttt atttttaatt ttctttcaaa
       61 tacttctagc tagagtattt ttacaacaat taccaacaac aacaacaaac aacaacaaca
      121 ttacatttta cattctacaa ctacagccat ggaatcccct atactaggtt attggaaaat
      181 taagggcctt gtgcaaccca ctcgacttct tttggaatat cttgaagaaa aatatgaaga
      241 gcatttgtat gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg aattgggttt
      301 ggagtttccc aatcttcctt attatattga tggtgatgtt aaattaacac agtctatggc
      361 catcatacgt tatatagctg acaagcacaa catgttgggt ggttgtccaa aagagcgtgc
      421 agagatttca atgcttgaag gagcggtttt ggatattaga tacggtgttt cgagaattgc
      481 atatagtaaa gactttgaaa ctctcaaagt tgattttctt agcaagctac ctgaaatgct
      541 gaaaatgttc gaagatcgtt tatgtcataa aacatattta aatggtgatc atgtaaccca
      601 tcctgacttc atgttgtatg acgctcttga tgttgtttta tacatggacc caatgtgcct
      661 ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt gaagctatcc cacaaattga
      721 taagtacttg aaatccagca agtatatagc atggcctttg cagggctggc aagccacgtt
      781 tggtggtggc gaccatcctc caaaatcgga tctggaagtt ctgttccagg ggcccgcgat
      841 cgccatggaa taagtaaagg aatcacatgg cacaggttat caacacgttt gacggggttg
      901 cggattatct tcagacatat cataagctac ctgataatta cattacaaaa tcagaagcac
      961 aagccctcgg ctgggtggca tcaaaaggga accttgcaga cgtcgctccg gggaaaagca
     1021 tcggcggaga catcttctca aacagggaag gcaaactccc gggcaaaagc ggacgaacat
     1081 ggcgtgaagc ggatattaac tatacatcag gcttcagaaa ttcagaccgg attctttact
     1141 caagcgactg gctgatttac aaaacaacgg accattatca gacctttaca aaaatcagat
     1201 aattaggcac cccaggcttt acactttatg ctttcggctc gtataatgtg tggattttga
     1261 gttaggatcc gtcgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg
     1321 gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt
     1381 cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga
     1441 ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt gcccgcctga
     1501 tgaatgctca tccggaattc cgtatggcaa tgaaagacgg tgagctggtg atatgggata
     1561 gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga
     1621 gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt
     1681 acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtctcag
     1741 ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct
     1801 tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc
     1861 tggcgattca ggttcatcat gccgtttgtg atggcttcca tgtcggcaga atgcttaatg
     1921 aattacaaca gtactgcgat gagtggcagg gcggggcgta atgtttaaac gaattcgagc
     1981 tcggtacccg gggatcctct agagtcgacc tgcaggcatg caagctgatc cggctgctaa
     2041 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcaaacta
     2101 gtttacggct agcgccggat gcgacgccgg tcgcgtctta tccggccttc ctatatcagg
     2161 ctgtgtttaa gacgccgccg cttcgcccaa atccttatgc cggttcgacg gctggacaaa
     2221 atactgttta tcttcccagc gcaggcaggt taatgtacca ccccagcagc agccggtatc
     2281 cagcgcgtat ataccttccg gcgtaccttt gccctccagc gatgcccagt gaccaaaggc
     2341 gatgctgtat tcttcagcga cagggccagg aatcgcaaac cacggtttca gtggggcagg
     2401 ggcctcttcc ggcgattctt actagctagt atgcataggt gctgaaatat aaagtttgtg
     2461 tttctaaaac acacgtggta cgtacgataa cgtacagtgt ttttccctcc acttaaatcg
     2521 aagggtagtg tcttggagcg cgcggagtaa acatatatgg ttcatatatg tccgtaggca
     2581 cgtaaaaaaa gcgagggatt cgaattcccc cggaaccccc ggttggggcc cacgcctcga
     2641 tcgagcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gcttggcgta atcatggtca
     2701 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     2761 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     2821 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     2881 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     2941 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3001 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3061 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3121 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3181 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3241 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3301 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3361 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3421 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3481 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     3541 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     3601 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     3661 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     3721 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     3781 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3841 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3901 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3961 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4021 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4081 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4141 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4201 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4261 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4321 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4381 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4441 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4501 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4561 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4621 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4681 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4741 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4801 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     4861 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     4921 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     4981 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     5041 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
     5101 catatcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     5161 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     5221 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     5281 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     5341 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatctggcta gcgatgaccc
     5401 tgctgattgg ttcgctgacc atttccgggg tgcggaacgg cgttaccaga aactcagaag
     5461 gttcgtccaa ccaaaccgac tctgacggca gtttacgaga gagatgatag ggtctgcttc
     5521 agtaagccag atgctacaca attaggcttg tacatattgt cgttagaacg cggctacaat
     5581 taatacataa ccttatgtat catacacata cg
//
