LOCUS       pET-32_Ek_LIC.        5917 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for ligation-independent cloning (LIC) to express 
            thioredoxin-tagged proteins with an enterokinase site.
ACCESSION   .
VERSION     .
KEYWORDS    pET-32 Ek_LIC.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5917)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5917)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     For LIC, linearize with BseRI and treat with T4 DNA polymerase plus 
            dTTP.
FEATURES             Location/Qualifiers
     source          1..5917
                     /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(236..250)
                     /label=enterokinase site
                     /note="enterokinase recognition and cleavage site"
     CDS             complement(266..310)
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     CDS             complement(317..334)
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
     CDS             complement(344..361)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(383..709)
                     /label=TrxA
                     /note="E. coli thioredoxin"
     RBS             complement(717..739)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(754..778)
                     /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(779..797)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1110..1187
                     /label=lacI promoter
     CDS             1188..2267
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2283..2304
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3079..3267
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3372..3514
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3700..4288)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4462..5319)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5320..5424)
                     /label=AmpR promoter
     rep_origin      complement(5451..5906)
                     /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 ccatggttga ggagaagccc gggctcttgt
      241 cgtcgtcatc ggtacccaga tctgggctgt ccatgtgctg gcgttcgaat ttagcagcag
      301 cggtttcttt cataccagaa ccgcgtggca ccagaccaga agaatgatga tgatgatggt
      361 gcatatggcc agaaccagaa ccggccaggt tagcgtcgag gaactctttc aactgacctt
      421 tagacagtgc acccactttg gttgccgcca cttcaccgtt tttgaacagc agcagagtcg
      481 ggataccacg gatgccatat ttcggcgcag tgccagggtt ttgatcgatg ttcagttttg
      541 caacggtcag tttgccctga tattcgtcag cgatttcatc cagaatcggg gcgatcattt
      601 tgcacggacc gcaccactct gcccagaaat cgacgaggat cgccccgtcc gctttgagta
      661 catccgtgtc aaaactgtcg tcagtcaggt gaataatttt atcgctcata tgtatatctc
      721 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc
      781 tatagtgagt cgtattaatt tcgcgggatc gagatcgatc tcgatcctct acgccggacg
      841 catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat
      901 caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg
      961 tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt
     1021 ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga
     1081 gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg
     1141 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
     1201 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     1261 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     1321 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     1381 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     1441 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     1501 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     1561 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     1621 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     1681 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     1741 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     1801 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     1861 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     1921 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     1981 gtgcggacat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
     2041 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     2101 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     2161 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     2221 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     2281 attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc
     2341 ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg
     2401 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc
     2461 ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga
     2521 atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag
     2581 aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg
     2641 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
     2701 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
     2761 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc
     2821 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct
     2881 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat
     2941 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc
     3001 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc
     3061 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt
     3121 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag
     3181 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg
     3241 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     3301 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     3361 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     3421 actatgcggc atcagagcag attgtactga gagtgcacca tatatgcggt gtgaaatacc
     3481 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
     3541 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3601 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3661 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3721 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3781 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3841 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3901 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3961 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4021 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4081 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4141 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4201 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4261 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4321 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4381 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4441 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4501 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4561 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4621 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4681 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4741 agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc
     4801 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4861 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4921 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4981 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     5041 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     5101 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5161 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5221 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5281 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5341 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5401 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg aaattgtaaa
     5461 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     5521 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     5581 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     5641 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     5701 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     5761 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     5821 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     5881 gcttaatgcg ccgctacagg gcgcgtccca ttcgcca
//
