LOCUS       sequence-209544-        5348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  yeast shuttle vector for insertion into yeast chromosomes in pBCSK 
            backbone. LoxP sites downstream of LEU2. For teaching purposes..
ACCESSION   .
VERSION     .
KEYWORDS    .
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  JOURNAL   Unpublished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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pBCSK_LEU2_II
FEATURES             Location/Qualifiers
     source          1..5348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     107..129
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     121..138
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     122..138
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     148..167
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        148..166
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        219..624
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             625..1719
                     /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(1860..1882)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1982..2001
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    complement(2037..2070)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(2125..2158)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2243..2263)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2243..2261)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2282..2298)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2282..2298)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2295..2317)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2306..2322
                     /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)."
     promoter        complement(join(2330..2336,2337..2354,2355..2360))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2375..2396
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2513..2530)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2684..3272)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2764..2783)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3492..3594
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3595..4254
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3661..3680)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4743..4847)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4873..5328)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5010..5031)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5222..5241
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
       61 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
      121 tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacg
      181 gcgcgcctgc agcatgctcg agctagcagc acgaggccaa ctgtgggaat actcaggtat
      241 cgtaagatgc aagagttcga atctcttagc aaccattatt tttttcctca acataacgag
      301 aacacacagg ggcgctatcg cacagaatca aattcgatga ctggaaattt tttgttaatt
      361 tcagaggtcg cctgacgcat ataccttttt caactgaaaa attgggagaa aaaggaaagg
      421 tgagagcgcc ggaaccggct tttcatatag aatagagaag cgttcatgac taaatgcttg
      481 catcacaata cttgaagttg acaatattat ttaaggacct attgtttttt ccaataggtg
      541 gttagcaatc gtcttacttt ctaacttttc ttacctttta catttcagca atatatatat
      601 atatttcaag gatataccat tctaatgtct gcccctaaga agatcgtcgt tttgccaggt
      661 gaccacgttg gtcaagaaat cacagccgaa gccattaagg ttcttaaagc tatttctgat
      721 gttcgttcca atgtcaagtt cgatttcgaa aatcatttaa ttggtggtgc tgctatcgat
      781 gctacaggtg ttccacttcc agatgaggcg ctggaagcct ccaagaaggc tgatgccgtt
      841 ttgttaggtg ctgtgggtgg tcctaaatgg ggtacaggta gtgttagacc tgaacaaggt
      901 ttactaaaaa tccgtaaaga acttcaattg tacgccaact taagaccatg taactttgca
      961 tccgactctc ttttagactt atctccaatc aagccacaat ttgctaaagg tactgacttc
     1021 gttgttgtca gagaattagt gggaggtatt tactttggta agagaaagga agacgatggt
     1081 gatggtgtcg cttgggatag tgaacaatac accgttccag aagtgcaaag aatcacaaga
     1141 atggccgctt tcatggccct acaacatgag ccaccattgc ctatttggtc cttggataaa
     1201 gctaatgttt tggcctcttc aagattatgg agaaaaactg tggaggaaac catcaagaac
     1261 gaatttccta cattgaaggt tcaacatcaa ttgattgatt ctgccgccat gatcctagtt
     1321 aagaacccaa cccacctaaa tggtattata atcaccagca acatgtttgg tgatatcatc
     1381 tccgatgaag cctccgttat cccaggttcc ttgggtttgt tgccatctgc gtccttggcc
     1441 tctttgccag acaagaacac cgcatttggt ttgtacgaac catgccacgg ttctgctcca
     1501 gatttgccaa agaataaggt caaccctatc gccactatct tgtctgctgc aatgatgttg
     1561 aaattgtcat tgaacttgcc tgaagaaggt aaggccattg aagatgcagt taaaaaggtt
     1621 ttggatgcag gtatcagaac tggtgattta ggtggttcca acagtaccac cgaagtcggt
     1681 gatgctgtcg ccgaagaagt taagaaaatc cttgcttaaa aagattctct ttttttatga
     1741 tatttgtaca taaactttat aaatgaaatt cataatagaa acgacacgaa attacaaaat
     1801 ggaatatgtt catagggtag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     1861 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     1921 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     1981 gcggcatcag agcagattgt actgagagtg cagctggtac cgggcccccc ctcgaaataa
     2041 cttcgtatag catacattat acgaagttat ccatggttga tgaaagaata acgtattctt
     2101 tcatcaacta gcaggcctgt cgaaataact tcgtatagca tacattatac gaagttatcc
     2161 atggttgatg aaagaataac gtattctttc atcaactagt tctagagcgg ccgccaccgc
     2221 ggtggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     2281 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     2341 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     2401 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     2461 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     2521 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     2581 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     2641 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2701 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2761 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2821 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2881 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2941 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3001 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3061 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3121 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3181 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3241 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3301 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3361 ggatcttcac ctagatcctt ttcgaccgaa taaatacctg tgacggaaga tcacttcgca
     3421 gaataaataa atcctggtgt ccctgttgat accgggaagc cctgggccaa cttttggcga
     3481 aaatgagacg ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca
     3541 ctaccgggcg tattttttga gttgtcgaga ttttcaggag ctaaggaagc taaaatggag
     3601 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
     3661 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
     3721 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
     3781 cttgcccgcc tgatgaatgc tcatccggaa ttacgtatgg caatgaaaga cggtgagctg
     3841 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
     3901 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     3961 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     4021 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     4081 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     4141 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
     4201 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt
     4261 taaggcagtt attggtgccc ttaaacgcct ggttgctacg cctgaataag tgataataag
     4321 cggatgaatg gcagaaattc gaaagcaaat tcgacccggt cgtcggttca gggcagggtc
     4381 gttaaatagc cgcttatgtc tattgctggt ttaccggttt attgactacc ggaagcagtg
     4441 tgaccgtgtg cttctcaaat gcctgaggcc agtttgctca ggctctcccc gtggaggtaa
     4501 taattgacga tatgatcctt tttttctgat caaaaagtgc tcatcattgg aaaacgttct
     4561 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4621 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4681 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4741 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4801 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4861 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     4921 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     4981 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     5041 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     5101 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     5161 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     5221 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     5281 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat
     5341 tcaggctg
//
