LOCUS       pET-32_Xa_LIC.        5926 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for ligation-independent cloning (LIC) to express 
            thioredoxin-tagged proteins with a Factor Xa site.
ACCESSION   .
VERSION     .
KEYWORDS    pET-32 Xa_LIC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5926)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5926)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     For LIC, linearize with BseRI and treat with T4 DNA polymerase plus 
            dCTP.
FEATURES             Location/Qualifiers
     source          1..5926
                     /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"
     misc_feature    158..216
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             complement(233..244)
                     /label=Factor Xa site
                     /note="Factor Xa recognition and cleavage site"
     CDS             complement(275..319)
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     CDS             complement(326..343)
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
     CDS             complement(353..370)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(392..718)
                     /label=TrxA
                     /note="E. coli thioredoxin"
     RBS             complement(726..748)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(763..787)
                     /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(788..806)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1119..1196
                     /label=lacI promoter
     CDS             1197..2276
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2292..2313
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3088..3276
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3381..3523
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3709..4297)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4471..5328)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5329..5433)
                     /label=AmpR promoter
     rep_origin      complement(5460..5915)
                     /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 gcaagcttgt
      181 cgacggagct cgaattcgga tccgatatcg ccatggccag aggagagtta gagcgaccct
      241 caataccgga gccaccaccg gtacccagat ctgggctgtc catgtgctgg cgttcgaatt
      301 tagcagcagc ggtttctttc ataccagaac cgcgtggcac cagaccagaa gaatgatgat
      361 gatgatggtg catatggcca gaaccagaac cggccaggtt agcgtcgagg aactctttca
      421 actgaccttt agacagtgca cccactttgg ttgccgccac ttcaccgttt ttgaacagca
      481 gcagagtcgg gataccacgg atgccatatt tcggcgcagt gccagggttt tgatcgatgt
      541 tcagttttgc aacggtcagt ttgccctgat attcgtcagc gatttcatcc agaatcgggg
      601 cgatcatttt gcacggaccg caccactctg cccagaaatc gacgaggatc gccccgtccg
      661 ctttgagtac atccgtgtca aaactgtcgt cagtcaggtg aataatttta tcgctcatat
      721 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca
      781 caattcccct atagtgagtc gtattaattt cgcgggatcg agatcgatct cgatcctcta
      841 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat
      901 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt
      961 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca
     1021 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct
     1081 aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa
     1141 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     1201 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     1261 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     1321 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     1381 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     1441 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     1501 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     1561 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     1621 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     1681 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     1741 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     1801 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     1861 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     1921 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     1981 tgcgcgttgg tgcggacatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     2041 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     2101 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     2161 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     2221 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     2281 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc
     2341 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc
     2401 gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg
     2461 gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg
     2521 gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt
     2581 ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg
     2641 ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa
     2701 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca
     2761 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg
     2821 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
     2881 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc
     2941 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca
     3001 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac
     3061 agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg
     3121 gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg
     3181 gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc
     3241 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     3301 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     3361 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
     3421 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atatgcggtg
     3481 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     3541 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3601 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3661 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3721 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3781 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3841 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3901 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3961 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4021 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4081 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4141 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4201 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4261 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4321 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4381 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4441 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4501 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4561 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4621 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4681 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4741 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc
     4801 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4861 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4921 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4981 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5041 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5101 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5161 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5221 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5281 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5341 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5401 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5461 aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     5521 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     5581 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     5641 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     5701 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     5761 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     5821 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     5881 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgcca
//
