LOCUS       Exported                5348 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  yeast shuttle vector for insertion into yeast chromosomes in pBCSK 
            backbone. LoxP sites upstream of LEU2. For teaching purposes.
ACCESSION   .
VERSION     .
KEYWORDS    pBCSK_LEU2_I
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5348)
  AUTHORS   Colin Logie
  TITLE     Logie Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5348)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5348
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    92..113
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        128..158
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    166..182
                     /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)."
     primer_bind     171..193
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     190..206
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     190..206
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     225..245
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        227..245
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(330..363)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(418..451)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        484..889
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             890..1984
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(2125..2147)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2247..2266
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2321..2340)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2322..2340)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2350..2367)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2350..2366)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2359..2381)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2508..2963
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2595..2614)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2805..2826
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2989..3093
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3582..4241)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4156..4175
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4242..4344)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      4564..5152
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5053..5072
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5306..5323
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
       61 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
      121 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
      181 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
      241 aaagggaaca aaagctggag ctccaccgcg gtggcggccg ctctagaact agttgatgaa
      301 agaatacgtt attctttcat caaccatgga taacttcgta taatgtatgc tatacgaagt
      361 tatttcgaca ggcctgctag ttgatgaaag aatacgttat tctttcatca accatggata
      421 acttcgtata atgtatgcta tacgaagtta tttcgagggg gggcccggta ccagcacgag
      481 gccaactgtg ggaatactca ggtatcgtaa gatgcaagag ttcgaatctc ttagcaacca
      541 ttattttttt cctcaacata acgagaacac acaggggcgc tatcgcacag aatcaaattc
      601 gatgactgga aattttttgt taatttcaga ggtcgcctga cgcatatacc tttttcaact
      661 gaaaaattgg gagaaaaagg aaaggtgaga gcgccggaac cggcttttca tatagaatag
      721 agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat attatttaag
      781 gacctattgt tttttccaat aggtggttag caatcgtctt actttctaac ttttcttacc
      841 ttttacattt cagcaatata tatatatatt tcaaggatat accattctaa tgtctgcccc
      901 taagaagatc gtcgttttgc caggtgacca cgttggtcaa gaaatcacag ccgaagccat
      961 taaggttctt aaagctattt ctgatgttcg ttccaatgtc aagttcgatt tcgaaaatca
     1021 tttaattggt ggtgctgcta tcgatgctac aggtgttcca cttccagatg aggcgctgga
     1081 agcctccaag aaggctgatg ccgttttgtt aggtgctgtg ggtggtccta aatggggtac
     1141 aggtagtgtt agacctgaac aaggtttact aaaaatccgt aaagaacttc aattgtacgc
     1201 caacttaaga ccatgtaact ttgcatccga ctctctttta gacttatctc caatcaagcc
     1261 acaatttgct aaaggtactg acttcgttgt tgtcagagaa ttagtgggag gtatttactt
     1321 tggtaagaga aaggaagacg atggtgatgg tgtcgcttgg gatagtgaac aatacaccgt
     1381 tccagaagtg caaagaatca caagaatggc cgctttcatg gccctacaac atgagccacc
     1441 attgcctatt tggtccttgg ataaagctaa tgttttggcc tcttcaagat tatggagaaa
     1501 aactgtggag gaaaccatca agaacgaatt tcctacattg aaggttcaac atcaattgat
     1561 tgattctgcc gccatgatcc tagttaagaa cccaacccac ctaaatggta ttataatcac
     1621 cagcaacatg tttggtgata tcatctccga tgaagcctcc gttatcccag gttccttggg
     1681 tttgttgcca tctgcgtcct tggcctcttt gccagacaag aacaccgcat ttggtttgta
     1741 cgaaccatgc cacggttctg ctccagattt gccaaagaat aaggtcaacc ctatcgccac
     1801 tatcttgtct gctgcaatga tgttgaaatt gtcattgaac ttgcctgaag aaggtaaggc
     1861 cattgaagat gcagttaaaa aggttttgga tgcaggtatc agaactggtg atttaggtgg
     1921 ttccaacagt accaccgaag tcggtgatgc tgtcgccgaa gaagttaaga aaatccttgc
     1981 ttaaaaagat tctctttttt tatgatattt gtacataaac tttataaatg aaattcataa
     2041 tagaaacgac acgaaattac aaaatggaat atgttcatag ggtaggccct ttcgtctcgc
     2101 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     2161 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     2221 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcagct
     2281 gctagctcga gcatgctgca ggcgcgccgt acccaattcg ccctatagtg agtcgtatta
     2341 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     2401 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     2461 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     2521 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2581 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2641 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2701 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2761 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2821 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2881 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     2941 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     3001 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3061 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3121 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3181 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     3241 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     3301 ttttgatcag aaaaaaagga tcatatcgtc aattattacc tccacgggga gagcctgagc
     3361 aaactggcct caggcatttg agaagcacac ggtcacactg cttccggtag tcaataaacc
     3421 ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac cgacgaccgg
     3481 gtcgaatttg ctttcgaatt tctgccattc atccgcttat tatcacttat tcaggcgtag
     3541 caaccaggcg tttaagggca ccaataactg ccttaaaaaa attacgcccc gccctgccac
     3601 tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg
     3661 gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg
     3721 cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg
     3781 gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg
     3841 aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc
     3901 cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa
     3961 acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata
     4021 cgtaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac
     4081 ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg
     4141 ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg
     4201 gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct
     4261 gaaaatctcg acaactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag
     4321 ttggaacctc ttacgtgccg atcaacgtct cattttcgcc aaaagttggc ccagggcttc
     4381 ccggtatcaa cagggacacc aggatttatt tattctgcga agtgatcttc cgtcacaggt
     4441 atttattcgg tcgaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4501 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4561 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4621 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4681 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4741 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4801 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4861 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4921 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4981 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     5041 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     5101 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5161 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5221 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     5281 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     5341 aatacgca
//
