LOCUS       pET-52b(+)_3C_LI        5227 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with an HRV 3C site for ligation-independent 
            cloning (LIC) to express proteins tagged with Strep-Tag II.
ACCESSION   .
VERSION     .
KEYWORDS    pET-52b(+) 3C_LIC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5227)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5227)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     For LIC, linearize with SacI and KpnI and treat with T4 DNA 
            polymerase plus dTTP.
FEATURES             Location/Qualifiers
     source          1..5227
                     /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(113..139)
                     /label=9xHis
                     /note="9xHis affinity tag"
     CDS             complement(146..163)
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
     CDS             complement(209..232)
                     /label=HRV 3C site
                     /note="recognition and cleavage site for human rhinovirus
                     3C and PreScission proteases"
     CDS             complement(239..262)
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
     CDS             complement(269..271)
                     /codon_start=1
                     /product="start"
                     /label=start
                     /note="ATG"
                     /translation="M"
     RBS             complement(278..300)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(315..339)
                     /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(340..358)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        671..748
                     /label=lacI promoter
     CDS             749..1828
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1844..1865
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2403..2591
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    2696..2838
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3024..3612)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        3693..3785
                     /label=AmpR promoter
     CDS             3786..4643
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      complement(4761..5216)
                     /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 cagcagccta ggttaattag tggtggtgat
      121 ggtgatgatg gtggtgatga gcggaactac cgcgtggcac cagagcgagc tctgcggccg
      181 cgtcgacttg tacaggatcc tggtacccgg gtccctgaaa gaggacttca agtgcaccct
      241 tttcgaactg cgggtggctc cagcttgcca tggtatatct ccttcttaaa gttaaacaaa
      301 attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat
      361 ttcgcgggat cgagatcgat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc
      421 ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg
      481 gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg
      541 gccgggggac tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc
      601 aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt
      661 cgagatcccg gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc
      721 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
      781 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
      841 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
      901 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
      961 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     1021 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     1081 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     1141 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     1201 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     1261 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     1321 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     1381 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     1441 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     1501 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggaca tctcggtagt
     1561 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     1621 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     1681 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     1741 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     1801 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     1861 ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct
     1921 tccggtgggc gcggggcatg actagcatga tcgtgctcct gtcgttgagg acccggctag
     1981 gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa
     2041 gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc
     2101 gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct
     2161 gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct
     2221 ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac
     2281 cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc
     2341 tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat
     2401 cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat
     2461 taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat
     2521 cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg
     2581 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     2641 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     2701 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     2761 gcagattgta ctgagagtgc accatatatg cggtgtgaaa taccgcacag atgcgtaagg
     2821 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     2881 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     2941 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3001 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3061 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3121 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3181 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3241 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3301 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3361 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3421 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     3481 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3541 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3601 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3661 aactcacgtt aagggatttt ggtcatgacc tatttgttta tttttctaaa tacattcaaa
     3721 tatgtatccg ctcatgagac aataaccctg ataaatgctt caatcatgat tgaaaaagga
     3781 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     3841 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     3901 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     3961 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4021 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4081 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4141 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4201 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4261 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4321 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4381 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4441 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4501 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4561 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4621 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     4681 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     4741 tcatgccatc gtgccgccag aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa
     4801 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     4861 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     4921 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     4981 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5041 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     5101 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     5161 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     5221 ttcgcca
//
