LOCUS       sequence-205847-        5359 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 5359)
  AUTHORS   Colin Logie
  TITLE     Logie Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5359)
  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            "Unpublished"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..5359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..129
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(617..1276)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1191..1210
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1277..1379)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      1599..2187
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2088..2107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2341..2358
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2475..2496
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2511..2516,2517..2534,2535..2541)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2549..2565
                     /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     2554..2576
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2573..2589
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2573..2589
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2608..2628
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2610..2628
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    2713..2746
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2801..2834
                     /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(2870..2889)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2989..3011
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3152..4246)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(4259..4663)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4715..4734)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4716..4734)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4744..4761)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4744..4760)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4753..4775)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4902..5357
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4989..5008)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5199..5220
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac ccttgataaa tgcttcaata atattgaaaa
      121 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
      181 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
      241 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
      301 tttcgccccg aagaacgttt tccaatgatg agcacttttg atcagaaaaa aaggatcata
      361 tcgtcaatta ttacctccac ggggagagcc tgagcaaact ggcctcaggc atttgagaag
      421 cacacggtca cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg
      481 gctatttaac gaccctgccc tgaaccgacg accgggtcga atttgctttc gaatttctgc
      541 cattcatccg cttattatca cttattcagg cgtagcaacc aggcgtttaa gggcaccaat
      601 aactgcctta aaaaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat
      661 taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg
      721 gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga
      781 agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg
      841 agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac
      901 acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc
      961 agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat
     1021 cccatatcac cagctcaccg tctttcattg ccatacgtaa ttccggatga gcattcatca
     1081 ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct
     1141 ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact
     1201 gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag
     1261 tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgacaac tcaaaaaata
     1321 cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa
     1381 cgtctcattt tcgccaaaag ttggcccagg gcttcccggt atcaacaggg acaccaggat
     1441 ttatttattc tgcgaagtga tcttccgtca caggtattta ttcggtcgaa aaggatctag
     1501 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1561 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1621 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1681 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1741 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1801 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1861 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1921 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     1981 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2041 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2101 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2161 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2221 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2281 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2341 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     2401 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     2461 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     2521 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     2581 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg
     2641 gagctccacc gcggtggcgg ccgctctaga actagttgat gaaagaatac gttattcttt
     2701 catcaaccat ggataacttc gtataatgta tgctatacga agttatttcg acaggcctgc
     2761 tagttgatga aagaatacgt tattctttca tcaaccatgg ataacttcgt ataatgtatg
     2821 ctatacgaag ttatttcgag ggggggcccg gtaccagctg cactctcagt acaatctgct
     2881 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     2941 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     3001 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctaccctatg
     3061 aacatattcc attttgtaat ttcgtgtcgt ttctattatg aatttcattt ataaagttta
     3121 tgtacaaata tcataaaaaa agagaatctt tttaagcaag gattttctta acttcttcgg
     3181 cgacagcatc accgacttcg gtggtactgt tggaaccacc taaatcacca gttctgatac
     3241 ctgcatccaa aaccttttta actgcatctt caatggcctt accttcttca ggcaagttca
     3301 atgacaattt caacatcatt gcagcagaca agatagtggc gatagggtca accttattct
     3361 ttggcaaatc tggagcagaa ccgtggcatg gttcgtacaa accaaatgcg gtgttcttgt
     3421 ctggcaaaga ggccaaggac gcagatggca acaaacccaa ggaacctggg ataacggagg
     3481 cttcatcgga gatgatatca ccaaacatgt tgctggtgat tataatacca tttaggtggg
     3541 ttgggttctt aactaggatc atggcggcag aatcaatcaa ttgatgttga accttcaatg
     3601 taggaaattc gttcttgatg gtttcctcca cagtttttct ccataatctt gaagaggcca
     3661 aaacattagc tttatccaag gaccaaatag gcaatggtgg ctcatgttgt agggccatga
     3721 aagcggccat tcttgtgatt ctttgcactt ctggaacggt gtattgttca ctatcccaag
     3781 cgacaccatc accatcgtct tcctttctct taccaaagta aatacctccc actaattctc
     3841 tgacaacaac gaagtcagta cctttagcaa attgtggctt gattggagat aagtctaaaa
     3901 gagagtcgga tgcaaagtta catggtctta agttggcgta caattgaagt tctttacgga
     3961 tttttagtaa accttgttca ggtctaacac tacctgtacc ccatttagga ccacccacag
     4021 cacctaacaa aacggcatca accttcttgg aggcttccag cgcctcatct ggaagtggga
     4081 cacctgtagc atcgatagca gcaccaccaa ttaaatgatt ttcgaaatcg aacttgacat
     4141 tggaacgaac atcagaaata gctttaagaa ccttaatggc ttcggctgtg atttcttgac
     4201 caacgtggtc acctggcaaa acgacgatct tcttaggggc agacataggg gcagacatta
     4261 gaatggtata tccttgaaat atatatatat attgctgaaa tgtaaaaggt aagaaaagtt
     4321 agaaagtaag acgattgcta accacctatt ggaaaaaaca ataggtcctt aaataatatt
     4381 gtcaacttca agtattgtga tgcaagcatt tagtcatgaa cgcttctcta ttctatatga
     4441 aaagccggtt ccggcctctc acctttcctt tttctcccaa tttttcagtt gaaaaaggta
     4501 tatgcgtcag gcgacctctg aaattaacaa aaaatttcca gtcatcgaat ttgattctgt
     4561 gcgatagcgc ccctgtgtgt tctcgttatg ttgaggaaaa aaataatggt tgctaagaga
     4621 ttcgaactct tgcatcttac gatacctgag tattcccaca gttggcctcg tgctgctagc
     4681 tcgagcatgc tgcaggcgcg ccgtacccaa ttcgccctat agtgagtcgt attacgcgcg
     4741 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     4801 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     4861 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     4921 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4981 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5041 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5101 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5161 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5221 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     5281 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5341 attaacgctt acaatttag
//
