LOCUS       40924_18301        7737 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pET15-MHL, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7737)
  AUTHORS   Loppnau P, Kozieradzki I, Arrowsmith C.
  TITLE     Expression vectors for high-throughput In-Fusion cloning
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7737)
  AUTHORS   Loppnau P, Kozieradzki I, Arrowsmith C.
  TITLE     Direct Submission
  JOURNAL   Submitted (26-FEB-2007) Structural Genomics Consortium, University 
            of Toronto, 100 College Street, Toronto, ON M5G1L5, Canada
REFERENCE   3  (bases 1 to 7737)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7737)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (26-FEB-2007) Structural Genomics Consortium, University of Toronto,
            100 College Street, Toronto, ON M5G1L5, Canada"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7737
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        104..208
                     /label=AmpR promoter
     CDS             209..1066
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1240..1828
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(2014..2156)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2261..2449)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(3727..3748)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3764..4843)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4844..4921)
                     /label=lacI promoter
     promoter        5230..5248
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5249..5273
                     /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             5288..5310
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5320..5337
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             5350..5370
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     protein_bind    complement(5398..5431)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5446..5891
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             5892..7310
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     misc_feature    7386..7400
                     /note="3'-LIC sequence; add complement to downstream PCR
                     primer"
     terminator      7478..7525
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(7700..7728)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2041 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc
     2101 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     2161 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     2221 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     2281 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     2341 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     2401 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     2461 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     2521 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     2581 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     2641 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     2701 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     2761 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     2821 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     2881 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga
     2941 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
     3001 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
     3061 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
     3121 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt
     3181 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt
     3241 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg
     3301 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat
     3361 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc
     3421 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     3481 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     3541 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     3601 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     3661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     3721 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3781 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3841 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     3901 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     3961 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     4021 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     4081 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     4141 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     4201 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     4261 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     4321 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     4381 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     4441 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     4501 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     4561 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     4621 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     4681 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     4741 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     4801 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     4861 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     4921 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     4981 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     5041 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     5101 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     5161 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     5221 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     5281 aaataatttt gtttaacttt aagaaggaga tataccatgc atcatcatca tcatcacagc
     5341 agcggcagag aaaacttgta tttccagggc catatgagtt ctcctcctga aagatccata
     5401 acttcgtata gcatacatta tacgaagtta tgcggccgcg acgtccacat atacctgccg
     5461 ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc
     5521 aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag
     5581 attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa
     5641 agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta
     5701 aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt
     5761 gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact
     5821 tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg
     5881 agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta
     5941 ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat
     6001 ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac
     6061 agcaaaaaaa tgaaaaatat caagttcctg agttcgattc gtccacaatt aaaaatatct
     6121 cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg
     6181 tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg
     6241 atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga
     6301 aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa
     6361 aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt
     6421 tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag
     6481 ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa
     6541 tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact
     6601 acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca
     6661 aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt
     6721 tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac
     6781 ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg
     6841 agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag
     6901 taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca
     6961 ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc
     7021 ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg
     7081 tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac
     7141 ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg
     7201 cagacaaaca atcaacgttt gcgcctagct tcctgctgaa catcaaaggc aagaaaacat
     7261 ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca
     7321 aaagaaaatg ccgatatcct attggcattg acgtcaggtg gcacttttcg aggagatcat
     7381 gcacatgatg acgaagcttg cggccgcact cgaggatccg gctgctaaca aagcccgaaa
     7441 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     7501 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat atcccgcaag
     7561 aggcccggca gtaccggcat aaccaagcct atgcctacag catccagggt gacggtgccg
     7621 aggatgacga tgagcgcatt gttagatttc atacacggtg cctgactgcg ttagcaattt
     7681 aactgtgata aactaccgca ttaaagctta tcgatgataa gctgtcaaac atgagaa
//
