LOCUS       pMCSG25.        5664 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial expression vector encoding an aspartic endopeptidase fused
            to a 6xHis-TEV leader.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG25.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5664)
  AUTHORS   Osipiuk J, Mulligan R, Bargassa M, Hamilton JE, Cunningham MA, 
            Joachimiak A.
  TITLE     Characterization of member of DUF1888 protein family, self-cleaving 
            and self-assembling endopeptidase.
  JOURNAL   J. Biol. Chem. 2012;287:19452-61.
  PUBMED    22493430
REFERENCE   2  (bases 1 to 5664)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5664)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Biol. 
            Chem."; date: "2012"; volume: "287"; pages: "19452-61"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SspI and 
            treat with T4 DNA polymerase plus dGTP.
FEATURES             Location/Qualifiers
     source          1..5664
                     /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)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(223..243)
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             complement(268..285)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(292..666)
                     /codon_start=1
                     /product="self-cleaving aspartic endopeptidase SO1698 from 
                     Shewanella oneidensis"
                     /note="aspartic endopeptidase"
                     /translation="MFAPQGLAQFIKVNVTLENGEPVFIYTDANGQVCQGDITVTQAGT
                     ITYLLNDQTLKGLKFVGVGFVTPFDGIIDAVTISSDGMLVQLVDLDKTPGTTKFQFVLS
                     NTANTLLVLSPDPQIINRPQN"
     RBS             complement(674..696)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(711..735)
                     /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(736..754)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1067..1144
                     /label=lacI promoter
     CDS             1145..2224
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2240..2261
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3036..3224
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3329..3471
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3657..4245)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4419..5276)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5277..5380)
                     /label=AmpR promoter
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccgttatcc acttccaata ttggattgga agtacaggtt
      241 ctcggtaccc agatctacac cagaagaatg atgatgatga tggtgcatat ggttttgtgg
      301 gcgattgata atttgaggat ctggacttaa aacgagtaat gtattagctg tattacttaa
      361 tacaaattgg aactttgtgg ttcctggtgt tttatctaaa tcgactagtt gtactaacat
      421 gccatcgcta ctaatggtaa ccgcatcgat aatgccatca aatggggtca caaagcctac
      481 accgacaaat tttaaacctt taagggtttg atcattgagt aagtaagtta ttgtgccagc
      541 ttgagtgacg gtgatatcac cttggcacac ttgaccattg gcatcggtat aaataaatac
      601 gggctcacca ttttctaggg ttacattgac ttttataaat tgagctaagc cttgtggtgc
      661 aaacatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
      721 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atcgatctcg
      781 atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc
      841 gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc
      901 gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc
      961 tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc
     1021 tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg
     1081 gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     1141 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     1201 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     1261 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     1321 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     1381 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     1441 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     1501 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     1561 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     1621 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     1681 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     1741 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     1801 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     1861 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     1921 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
     1981 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     2041 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     2101 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     2161 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     2221 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac
     2281 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta
     2341 tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag
     2401 cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt
     2461 cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca
     2521 ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca cgggtgcgca
     2581 tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc
     2641 agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga
     2701 cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga
     2761 agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct
     2821 gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct
     2881 ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat
     2941 gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc
     3001 ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc
     3061 ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc
     3121 tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt
     3181 accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     3241 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     3301 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
     3361 gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     3421 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     3481 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3541 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3601 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3661 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3721 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3781 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3841 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3901 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3961 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4021 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4081 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4141 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4201 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4261 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4321 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4381 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4441 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4501 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4561 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4621 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4681 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc
     4741 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4801 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4861 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4921 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4981 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5041 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5101 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5161 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5221 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5281 ttcctttttc aaattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5341 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5401 cacctaaatt gtaagcgtta atgtgaacca tcaccctaat caagtttttt ggggtcgagg
     5461 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     5521 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     5581 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     5641 ctacagggcg cgtcccattc gcca
//
