LOCUS       40924_18471        7889 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pET32a-LIC, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7889)
  AUTHORS   Loppnau P, Kozieradzki I, Arrowsmith C.
  TITLE     Expression vectors for high-throughput In-Fusion cloning
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7889)
  AUTHORS   Loppnau P, Kozieradzki I, Arrowsmith C.
  TITLE     Direct Submission
  JOURNAL   Submitted (19-FEB-2007) Structural Genomics Consortium, University 
            of Toronto, 100 College Street, Toronto, ON M5G1L5, Canada
REFERENCE   3  (bases 1 to 7889)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7889)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (19-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..7889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        494..598
                     /label=AmpR promoter
     CDS             599..1456
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1630..2218
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(2404..2546)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2651..2839)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(3614..3635)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3651..4730)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4731..4808)
                     /label=lacI promoter
     promoter        5121..5139
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5140..5164
                     /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             5179..5201
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5209..5535
                     /label=TrxA
                     /note="E. coli thioredoxin"
     CDS             5557..5574
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             5584..5601
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
     CDS             5608..5652
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     CDS             5668..5688
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     protein_bind    complement(5716..5749)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5764..6209
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             6210..7628
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     misc_feature    7704..7718
                     /note="3'-cloning sequence; add complement to downstream
                     PCR primer"
     CDS             7733..7750
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      7817..7864
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
      601 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
      661 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
      721 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
      781 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      841 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      901 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      961 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     1021 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     1081 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     1141 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     1201 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     1261 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     1321 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     1381 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     1441 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     1501 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     1561 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1621 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1681 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1741 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     1801 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1861 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1921 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1981 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     2041 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     2101 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2161 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     2221 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2281 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     2341 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     2401 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg
     2461 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     2521 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     2581 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     2641 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     2701 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     2761 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     2821 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     2881 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     2941 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     3001 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     3061 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     3121 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     3181 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     3241 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     3301 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     3361 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     3421 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     3481 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     3541 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     3601 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     3661 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3721 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     3781 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     3841 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     3901 tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta
     3961 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     4021 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     4081 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     4141 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     4201 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     4261 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     4321 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     4381 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     4441 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     4501 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     4561 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     4621 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     4681 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     4741 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     4801 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     4861 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     4921 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     4981 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     5041 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     5101 cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     5161 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat gagcgataaa
     5221 attattcacc tgactgacga cagttttgac acggatgtac tcaaagcgga cggggcgatc
     5281 ctcgtcgatt tctgggcaga gtggtgcggt ccgtgcaaaa tgatcgcccc gattctggat
     5341 gaaatcgctg acgaatatca gggcaaactg accgttgcaa aactgaacat cgatcaaaac
     5401 cctggcactg cgccgaaata tggcatccgt ggtatcccga ctctgctgct gttcaaaaac
     5461 ggtgaagtgg cggcaaccaa agtgggtgca ctgtctaaag gtcagttgaa agagttcctc
     5521 gacgctaacc tggccggttc tggttctggc catatgcacc atcatcatca tcattcttct
     5581 ggtctggtgc cacgcggttc tggtatgaaa gaaaccgctg ctgctaaatt cgaacgccag
     5641 cacatggaca gcccagatct gggtaccgaa aacttgtatt tccagagcca tatgagttct
     5701 cctcctgaaa gatccataac ttcgtatagc atacattata cgaagttatg cggccgcgac
     5761 gtccacatat acctgccgtt cactattatt tagtgaaatg agatattatg atattttctg
     5821 aattgtgatt aaaaaggcaa ctttatgccc atgcaacaga aactataaaa aatacagaga
     5881 atgaaaagaa acagatagat tttttagttc tttaggcccg tagtctgcaa atccttttat
     5941 gattttctat caaacaaaag aggaaaatag accagttgca atccaaacga gagtctaata
     6001 gaatgaggtc gaaaagtaaa tcgcgcgggt ttgttactga taaagcaggc aagacctaaa
     6061 atgtgtaaag ggcaaagtgt atactttggc gtcacccctt acatatttta ggtctttttt
     6121 tattgtgcgt aactaacttg ccatcttcaa acaggagggc tggaagaagc agaccgctaa
     6181 cacagtacat aaaaaaggag acatgaacga tgaacatcaa aaagtttgca aaacaagcaa
     6241 cagtattaac ctttactacc gcactgctgg caggaggcgc aactcaagcg tttgcgaaag
     6301 aaacgaacca aaagccatat aaggaaacat acggcatttc ccatattaca cgccatgata
     6361 tgctgcaaat ccctgaacag caaaaaaatg aaaaatatca agttcctgag ttcgattcgt
     6421 ccacaattaa aaatatctct tctgcaaaag gcctggacgt ttgggacagc tggccattac
     6481 aaaacgctga cggcactgtc gcaaactatc acggctacca catcgtcttt gcattagccg
     6541 gagatcctaa aaatgcggat gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa
     6601 cttctattga cagctggaaa aacgctggcc gcgtctttaa agacagcgac aaattcgatg
     6661 caaatgattc tatcctaaaa gaccaaacac aagaatggtc aggttcagcc acatttacat
     6721 ctgacggaaa aatccgttta ttctacactg atttctccgg taaacattac ggcaaacaaa
     6781 cactgacaac tgcacaagtt aacgtatcag catcagacag ctctttgaac atcaacggtg
     6841 tagaggatta taaatcaatc tttgacggtg acggaaaaac gtatcaaaat gtacagcagt
     6901 tcatcgatga aggcaactac agctcaggcg acaaccatac gctgagagat cctcactacg
     6961 tagaagataa aggccacaaa tacttagtat ttgaagcaaa cactggaact gaagatggct
     7021 accaaggcga agaatcttta tttaacaaag catactatgg caaaagcaca tcattcttcc
     7081 gtcaagaaag tcaaaaactt ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg
     7141 gcgctctcgg tatgattgag ctaaacgatg attacacact gaaaaaagtg atgaaaccgc
     7201 tgattgcatc taacacagta acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg
     7261 gcaaatggta cctgttcact gactcccgcg gatcaaaaat gacgattgac ggcattacgt
     7321 ctaacgatat ttacatgctt ggttatgttt ctaattcttt aactggccca tacaagccgc
     7381 tgaacaaaac tggccttgtg ttaaaaatgg atcttgatcc taacgatgta acctttactt
     7441 actcacactt cgctgtacct caagcgaaag gaaacaatgt cgtgattaca agctatatga
     7501 caaacagagg attctacgca gacaaacaat caacgtttgc gcctagcttc ctgctgaaca
     7561 tcaaaggcaa gaaaacatct gttgtcaaag acagcatcct tgaacaagga caattaacag
     7621 ttaacaaata aaaacgcaaa agaaaatgcc gatatcctat tggcattgac gtcaggtggc
     7681 acttttcgag gagatcatgc acatgatgac gaagcttgcg gccgcactcg agcaccacca
     7741 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     7801 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     7861 tttgctgaaa ggaggaacta tatccggat
//
