LOCUS       Exported                6980 bp DNA     circular SYN 12-NOV-2024
DEFINITION  Expression vector pCW-LIC, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6980)
  AUTHORS   Loppnau P, Kozieradzki I, Arrowsmith C.
  TITLE     Expression vectors for high-throughput In-Fusion cloning
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6980)
  AUTHORS   Loppnau P, Kozieradzki I, Arrowsmith C.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-FEB-2007) Structural Genomics Consortium, University 
            of Toronto, 100 College Street, Toronto, ON M5G1L5, Canada
REFERENCE   3  (bases 1 to 6980)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6980)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 6980)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (27-FEB-2007) Structural Genomics Consortium, University of Toronto,
            100 College Street, Toronto, ON M5G1L5, Canada"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6980
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..465
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        492..596
                     /label=AmpR promoter
     CDS             597..1454
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1628..2216
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(2402..2544)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2953..3126)
                     /label=lacZ-alpha
                     /note="LacZ-alpha fragment of beta-galactosidase"
     protein_bind    complement(3146..3162)
                     /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(3170..3200)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3215..3236)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3252..4331)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4332..4409)
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     protein_bind    4461..4482
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        4497..4527
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     protein_bind    4535..4551
                     /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        4606..4634
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    4640..4660
                     /label=lac repressor protein binding site
                     /bound_moiety="lac repressor protein"
     protein_bind    4642..4658
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     regulatory      4701..4729
                     /label=Ptac
                     /note="Ptac"
                     /regulatory_class="promoter"
     promoter        4701..4729
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     regulatory      4701..4706
                     /label=derived from trp promoter
                     /note="derived from trp promoter"
                     /regulatory_class="minus_35_signal"
     regulatory      4723..4729
                     /label=derived from lacUV5 promoter
                     /note="derived from lacUV5 promoter"
                     /regulatory_class="minus_10_signal"
     protein_bind    4735..4755
                     /label=lac repressor protein binding site
                     /bound_moiety="lac repressor protein"
     protein_bind    4737..4753
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     misc_feature    4784..4798
                     /note="5'-cloning sequence; add to upstream PCR primer"
     protein_bind    complement(4826..4859)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4874..5319
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             5320..6738
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSCPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     misc_feature    6814..6828
                     /note="3'-cloning sequence; add complement to downstream
                     PCR primer"
     regulatory      6862..6889
                     /label=trp operon transcription termination sequence
                     /note="trp operon transcription termination sequence"
                     /regulatory_class="terminator"
ORIGIN
        1 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatcaggt ggcacttttc
      481 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
      541 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
      601 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
      661 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
      721 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
      781 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
      841 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
      901 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
      961 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     1021 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     1081 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     1141 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     1201 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     1261 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     1321 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     1381 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     1441 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     1501 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     1561 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     1621 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     1681 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     1741 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     1801 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     1861 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     1921 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     1981 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2041 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     2101 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2161 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2221 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2281 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2341 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     2401 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg
     2461 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     2521 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     2581 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     2641 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagaacgc catcaaaaat
     2701 aattcgcgtc tggccttcct gtagccagct ttcatcaaca ttaaatgtga gcgagtaaca
     2761 acccgtcgga ttctccgtgg gaacaaacgg cggattgacc gtaatgggat aggttacgtt
     2821 ggtgtagatg ggcgcatcgt aaccgtgcat ctgccagttt gaggggacga cgacagtatc
     2881 ggcctcagga agatcgcact ccagccagct ttccggcacc gcttctggtg ccggaaacca
     2941 ggcaaagcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     3001 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     3061 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat ccgtaatcat
     3121 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3181 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3241 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3301 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     3361 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     3421 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttgacggc
     3481 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     3541 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     3601 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     3661 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     3721 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     3781 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     3841 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     3901 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     3961 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     4021 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     4081 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     4141 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     4201 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     4261 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     4321 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     4381 cgaaaggttt tgcaccattc gatggtgtcc tggcacgaca ggtttcccga ctggaaagcg
     4441 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc ccaggcttta
     4501 cactttatgc ttccggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca
     4561 ggaaacagga tcgatccatc gatgagctta ctccccatcc ccctgttgac aattaatcat
     4621 cggctcgtat aatgtgtgga attgtgagcg gataacaatt tcacacagga aacaggatca
     4681 gcttactccc catccccctg ttgacaatta atcatcggct cgtataatgt gtggaattgt
     4741 gagcggataa caatttcaca caggaaacag gatccatcga tgcttaggag gtcatatgat
     4801 tatgagttct cctcctgaaa gatccataac ttcgtatagc atacattata cgaagttatg
     4861 cggccgcgac gtccacatat acctgccgtt cactattatt tagtgaaatg agatattatg
     4921 atattttctg aattgtgatt aaaaaggcaa ctttatgccc atgcaacaga aactataaaa
     4981 aatacagaga atgaaaagaa acagatagat tttttagttc tttaggcccg tagtctgcaa
     5041 atccttttat gattttctat caaacaaaag aggaaaatag accagttgca atccaaacga
     5101 gagtctaata gaatgaggtc gaaaagtaaa tcgcgcgggt ttgttactga taaagcaggc
     5161 aagacctaaa atgtgtaaag ggcaaagtgt atactttggc gtcacccctt acatatttta
     5221 ggtctttttt tattgtgcgt aactaacttg ccatcttcaa acaggagggc tggaagaagc
     5281 agaccgctaa cacagtacat aaaaaaggag acatgaacga tgaacatcaa aaagtttgca
     5341 aaacaagcaa cagtattaac ctttactacc gcactgctgg caggaggcgc aactcaagcg
     5401 tttgcgaaag aaacgaacca aaagccatat aaggaaacat acggcatttc ccatattaca
     5461 cgccatgata tgctgcaaat ccctgaacag caaaaaaatg aaaaatatca agttcctgaa
     5521 ttcgattcgt ccacaattaa aaatatctct tctgcaaaag gcctggacgt ttgggacagc
     5581 tgcccattac aaaacgctga cggcactgtc gcaaactatc acggctacca catcgtcttt
     5641 gcattagccg gagatcctaa aaatgcggat gacacatcga tttacatgtt ctatcaaaaa
     5701 gtcggcgaaa cttctattga cagctggaaa aacgctggcc gcgtctttaa agacagcgac
     5761 aaattcgatg caaatgattc tatcctaaaa gaccaaacac aagaatggtc aggttcagcc
     5821 acatttacat ctgacggaaa aatccgttta ttctacactg atttctccgg taaacattac
     5881 ggcaaacaaa cactgacaac tgcacaagtt aacgtatcag catcagacag ctctttgaac
     5941 atcaacggtg tagaggatta taaatcaatc tttgacggtg acggaaaaac gtatcaaaat
     6001 gtacagcagt tcatcgatga aggcaactac agctcaggcg acaaccatac gctgagagat
     6061 cctcactacg tagaagataa aggccacaaa tacttagtat ttgaagcaaa cactggaact
     6121 gaagatggct accaaggcga agaatcttta tttaacaaag catactatgg caaaagcaca
     6181 tcattcttcc gtcaagaaag tcaaaaactt ctgcaaagcg ataaaaaacg cacggctgag
     6241 ttagcaaacg gcgctctcgg tatgattgag ctaaacgatg attacacact gaaaaaagtg
     6301 atgaaaccgc tgattgcatc taacacagta acagatgaaa ttgaacgcgc gaacgtcttt
     6361 aaaatgaacg gcaaatggta cctgttcact gactcccgcg gatcaaaaat gacgattgac
     6421 ggcattacgt ctaacgatat ttacatgctt ggttatgttt ctaattcttt aactggccca
     6481 tacaagccgc tgaacaaaac tggccttgtg ttaaaaatgg atcttgatcc taacgatgta
     6541 acctttactt actcacactt cgctgtacct caagcgaaag gaaacaatgt cgtgattaca
     6601 agctatatga caaacagagg attctacgca gacaaacaat caacgtttgc gccaagcttc
     6661 ctgctgaaca tcaaaggcaa gaaaacatct gttgtcaaag acagcatcct tgaacaagga
     6721 caattaacag ttaacaaata aaaacgcaaa agaaaatgcc gatatcctat tggcattgac
     6781 gtcaggtggc acttttcgag gagatcatgc acatgatgac gaagcttatc gatgataagc
     6841 tgtcaaacat gagcagatct gagcccgcct aatgagcggg cttttttttc agatctgctt
     6901 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     6961 tttcttaggt gctcgtcaaa
//
