LOCUS       pET-30_Xa_LIC.        5448 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for ligation-independent cloning (LIC) to express 
            6xHis- and S-tagged proteins with a Factor Xa site.
ACCESSION   .
VERSION     .
KEYWORDS    pET-30 Xa_LIC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5448)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5448)
  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..5448
                     /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..219
                     /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(371..373)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(381..403)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(418..442)
                     /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(443..461)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        774..851
                     /label=lacI promoter
     CDS             852..1931
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1947..1968
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2743..2931
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3036..3178
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3364..3952)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             4074..4886
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      complement(4982..5437)
                     /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 catatgtata tctccttctt aaagttaaac aaaattattt ctagagggga
      421 attgttatcc gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc
      481 gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt
      541 tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct
      601 catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg
      661 cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact
      721 actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca
      781 tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
      841 gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
      901 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
      961 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
     1021 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
     1081 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
     1141 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
     1201 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
     1261 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
     1321 acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat
     1381 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
     1441 gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg
     1501 aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca
     1561 tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca
     1621 ttaccgagtc cgggctgcgc gttggtgcgg acatctcggt agtgggatac gacgataccg
     1681 aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg
     1741 ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc
     1801 agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg
     1861 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     1921 aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga
     1981 tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc
     2041 atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg
     2101 ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc
     2161 ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt
     2221 cccgccacca aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg
     2281 gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact
     2341 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg
     2401 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa
     2461 acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg
     2521 ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt
     2581 tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac
     2641 cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc
     2701 attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa
     2761 accgccctta acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc
     2821 aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat
     2881 gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     2941 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     3001 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     3061 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     3121 accatatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     3181 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3241 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3301 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3361 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3421 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3481 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3541 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3601 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3661 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3721 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3781 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     3841 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3901 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3961 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4021 ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     4081 atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt
     4141 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat
     4201 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     4261 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     4321 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     4381 ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     4441 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     4501 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     4561 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     4621 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     4681 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     4741 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     4801 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     4861 ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata catatttgaa
     4921 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4981 gaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     5041 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     5101 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     5161 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     5221 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     5281 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     5341 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     5401 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgcca
//
