LOCUS       40924_33237        7317 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNIC-NHT-CF, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7317)
  AUTHORS   Gileadi O, Burgess-Brown N, Loppnau P.
  TITLE     Vectors for Ligation-independent Cloning (LIC)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7317)
  AUTHORS   Gileadi O, Burgess-Brown N.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-DEC-2006) Structural Genomics Consortium, University 
            of Oxford, Botnar Research Centre, Oxford OX3 7LD, UK
REFERENCE   3  (bases 1 to 7317)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7317)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (27-DEC-2006) Structural Genomics Consortium, University of Oxford, 
            Botnar Research Centre, Oxford OX3 7LD, UK"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7317
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             134..154
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     promoter        170..615
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             616..2034
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     misc_feature    2081..2097
                     /note="sequence used for ligation-independent cloning; add
                     to downstream primer; the same sequence appears in a vector
                     fragment created by BsaI cleavage"
     CDS             2105..2128
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             2178..2195
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      2262..2309
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2346..2801
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(2897..3709)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      3831..4419
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4605..4747)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4852..5040)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(5815..5836)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5852..6931)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(6932..7009)
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatggag accgacgtcc acatatacct
      181 gccgttcact attatttagt gaaatgagat attatgatat tttctgaatt gtgattaaaa
      241 aggcaacttt atgcccatgc aacagaaact ataaaaaata cagagaatga aaagaaacag
      301 atagattttt tagttcttta ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa
      361 caaaagagga aaatagacca gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa
      421 agtaaatcgc gcgggtttgt tactgataaa gcaggcaaga cctaaaatgt gtaaagggca
      481 aagtgtatac tttggcgtca ccccttacat attttaggtc tttttttatt gtgcgtaact
      541 aacttgccat cttcaaacag gagggctgga agaagcagac cgctaacaca gtacataaaa
      601 aaggagacat gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt
      661 actaccgcac tgctggcagg aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag
      721 ccatataagg aaacatacgg catttcccat attacacgcc atgatatgct gcaaatccct
      781 gaacagcaaa aaaatgaaaa atataaagtt cctgagttcg attcgtccac aattaaaaat
      841 atctcttctg caaaaggcct ggacgtttgg gacagctggc cattacaaaa cactgacggc
      901 actgtcgcaa actatcacgg ctaccacatc gtctttgcat tagccggaga tcctaaaaat
      961 gcggatgaca catcgattta catgttctat caaaaagtcg gcgaaacttc tattgacagc
     1021 tggaaaaacg ctggccgcgt ctttaaagac agcgacaaat tcgatgcaaa tgattctatc
     1081 ctaaaagacc aaacacaaga atggtcaggt tcagccacat ttacatctga cggaaaaatc
     1141 cgtttattct acactgattt ctccggtaaa cattacggca aacaaacact gacaactgca
     1201 caagttaacg tatcagcatc agacagctct ttgaacatca acggtgtaga ggattataaa
     1261 tcaatctttg acggtgacgg aaaaacgtat caaaatgtac agcagttcat cgatgaaggc
     1321 aactacagct caggcgacaa ccatacgctg agagatcctc actacgtaga agataaaggc
     1381 cacaaatact tagtatttga agcaaacact ggaactgaag atggctacca aggcgaagaa
     1441 tctttattta acaaagcata ctatggcaaa agcacatcat tcttccgtca agaaagtcaa
     1501 aaacttctgc aaagcgataa aaaacgcacg gctgagttag caaacggcgc tctcggtatg
     1561 attgagctaa acgatgatta cacactgaaa aaagtgatga aaccgctgat tgcatctaac
     1621 acagtaacag atgaaattga acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg
     1681 ttcactgact cccgcggatc aaaaatgacg attgacggca ttacgtctaa cgatatttac
     1741 atgcttggtt atgtttctaa ttctttaact ggcccataca agccgctgaa caaaactggc
     1801 cttgtgttaa aaatggatct tgatcctaac gatgtaacct ttacttactc acacttcgct
     1861 gtacctcaag cgaaaggaaa caatgtcgtg attacaagct atatgacaaa cagaggattc
     1921 tacgcagaca aacaatcaac gtttgcgcct agcttcctgc tgaacatcaa aggcaagaaa
     1981 acatctgttg tcaaagacag catccttgaa caaggacaat taacagttaa caaataaaaa
     2041 cgcaaaagaa aatgccgata tcctattggc attgacggtc tccagtaaag gtggatacgg
     2101 atctgattac aaggatgacg acgataagtg aagatccgaa ttcgagctcc gtcgacaagc
     2161 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     2221 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     2281 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     2341 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2401 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2461 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2521 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2581 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2641 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2701 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2761 atttaacgcg aattttaaca aaatattaac gtttacaatt tcaggtggca cttttcgggg
     2821 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2881 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     2941 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     3001 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     3061 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     3121 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     3181 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     3241 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     3301 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     3361 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     3421 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     3481 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     3541 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     3601 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     3661 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     3721 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     3781 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3841 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3901 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3961 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     4021 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4081 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4141 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4201 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4261 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4321 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4381 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4441 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4501 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4561 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     4621 ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct
     4681 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4741 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4801 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4861 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4921 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4981 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     5041 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     5101 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     5161 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     5221 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     5281 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     5341 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5401 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5461 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     5521 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5581 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5641 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5701 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5761 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5821 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5881 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5941 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     6001 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     6061 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     6121 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     6181 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     6241 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     6301 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     6361 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     6421 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6481 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6541 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     6601 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     6661 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6721 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     6781 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     6841 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     6901 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     6961 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     7021 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     7081 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     7141 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     7201 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     7261 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
