LOCUS       sequence-108547-        5938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Episomal E. coli/S. cerevisiae shuttle vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5938)
  AUTHORS   Gnugge R, Liphardt T, Rudolf F
  TITLE     A shuttle vector series for precise genetic engineering of 
            Saccharomyces cerevisiae.
  JOURNAL   Yeast. 2015 Dec 9. doi: 10.1002/yea.3144.
  PUBMED    26647923
REFERENCE   2  (bases 1 to 5938)
  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: "Yeast. 2015
            Dec 9. doi: 10.1002/yea.3144."
COMMENT     Sequence Label: pRG225
FEATURES             Location/Qualifiers
     source          1..5938
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      7..1349
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(970..1017)
                     /label=FRT (mutant)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FRT site with a mutation in the core sequence (Azam 
                     et al., 1997)"
     protein_bind    complement(970..1017)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(1646..2740)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFQNHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(2741..3146)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     3342..3364
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3356..3373
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3357..3373
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3380..3399
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3380..3398
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     3507..3523
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3507..3523
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3557..3573)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3558..3574)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(3672..3690)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3711..3727)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3711..3727)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3724..3746)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3735..3751
                     /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     complement(4010..4027)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4181..4769)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4261..4280)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4948..5739,5740..5808))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5571..5590
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5809..5913)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcaacg aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta
       61 atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg
      121 ctattttacc aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc aacgcgagag
      181 cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga
      241 gagcgctatt ttaccaacaa agaatctata cttctttttt gttctacaaa aatgcatccc
      301 gagagcgcta tttttctaac aaagcatctt agattacttt ttttctcctt tgtgcgctct
      361 ataatgcagt ctcttgataa ctttttgcac tgtaggtccg ttaaggttag aagaaggcta
      421 ctttggtgtc tattttctct tccataaaaa aagcctgact ccacttcccg cgtttactga
      481 ttactagcga agctgcgggt gcattttttc aagataaagg catccccgat tatattctat
      541 accgatgtgg attgcgcata ctttgtgaac agaaagtgat agcgttgatg attcttcatt
      601 ggtcagaaaa ttatgaacgg tttcttctat tttgtctcta tatactacgt ataggaaatg
      661 tttacatttt cgtattgttt tcgattcact ctatgaatag ttcttactac aatttttttg
      721 tctaaagagt aatactagag ataaacataa aaaatgtaga ggtcgagttt agatgcaagt
      781 tcaaggagcg aaaggtggat gggtaggtta tatagggata tagcacagag atatatagca
      841 aagagatact tttgagcaat gtttgtggaa gcggtattcg caatatttta gtagctcgtt
      901 acagtccggt gcgtttttgg ttttttgaaa gtgcgtcttc agagcgcttt tggttttcaa
      961 aagcgctctg aagttcctat actttttaga gaataggaac ttcggaatag gaacttcaaa
     1021 gcgtttccga aaacgagcgc ttccgaaaat gcaacgcgag ctgcgcacat acagctcact
     1081 gttcacgtcg cacctatatc tgcgtgttgc ctgtatatat atatacatga gaagaacggc
     1141 atagtgcgtg tttatgctta aatgcgtact tatatgcgtc tatttatgta ggatgaaagg
     1201 tagtctagta cctcctgtga tattatccca ttccatgcgg ggtatcgtat gcttccttca
     1261 gcactaccct ttagctgttc tatatgctgc cactcctcaa ttggattagt ctcatccttc
     1321 aatgctatca tttcctttga tattggatca tattaattaa ctctggcgcg cctaactttt
     1381 tgtgtggtgc cctcctcctt gtcaatatta atgttaaagt gcaattcttt ttccttatca
     1441 cgttgagcca ttagtatcaa tttgcttacc tgtattcctt tacatcctcc tttttctcct
     1501 tcttgataaa tgtatgtaga ttgcgtatat agtttcgtct accctatgaa catattccat
     1561 tttgtaattt cgtgtcgcat atgtttctat tatgaatttc atttataaag tttatgtaca
     1621 aatatcataa aaaaagagaa tctttttaag caaggatttt cttaacttct tcggcgacag
     1681 catcaccgac ttcggtggta ctgttggaac cacctaaatc accagttctg atacctgcat
     1741 ccaaaacctt tttaactgca tcttcaatgg ccttaccttc ttcaggcaag ttcaatgaca
     1801 atttcaacat cattgcagca gacaagatag tggcgatagg gttgacctta ttctttggca
     1861 aatctggagc agaaccgtgg catggttcgt acaaaccaaa tgcggtgttc ttgtctggca
     1921 aagaggccaa ggacgcagat ggcaacaaac ccaaggaacc tgggataacg gaggcttcat
     1981 cggagatgat gtcaccaaac atgttgctgg tgattataat accatttagg tgggttgggt
     2041 tcttaactag gatcatggcg gcagaatcaa tcaattgatg ttgaaccttc aatgtaggaa
     2101 attcgttctt gatggtttcc tccacagttt ttctccataa tcttgaagag gccaaaacat
     2161 tagctttatc caaggaccaa ataggcaatg gtggctcatg ttgtagggcc atgaaagcgg
     2221 ccattcttgt gattctttgc acttctggaa cggtgtattg ttcactatcc caagcgacac
     2281 catcaccatc gtcttccttt ctcttaccaa agtaaatacc tcccactaat tctctgacaa
     2341 caacgaagtc agtaccttta gcaaattgtg gcttgattgg agataagtct aaaagagagt
     2401 cggatgcaaa gttacatggt cttaagttgg cgtacaattg aagttcttta cggattttta
     2461 gtaaaccttg ttcaggtcta acactaccag taccccattt aggaccaccc acagcaccta
     2521 acaaaacggc atcagccttc ttggaggctt ccagcgcctc atctggaagt ggaacacctg
     2581 tagcatcgat agcagcacca ccaattaaat gattttggaa atcgaacttg acattggaac
     2641 gaacatcaga aatagcttta agaaccttaa tggcttcggc tgtgatttct tgaccaacgt
     2701 ggtcacctgg caaaacgacg atcttcttag gggcagacat tagaatggta tatccttgaa
     2761 atatatatat atattgctga aatgtaaaag gtaagaaaag ttagaaagta agacgattgc
     2821 taaccaccta ttggaaaaaa caataggtcc ttaaataata ttgtcaactt caagtattgt
     2881 gatgcaagca tttagtcatg aacgcttctc tattctatat gaaaagccgg ttccggcgct
     2941 ctcacctttc ctttttctcc caatttttca gttgaaaaag gtatatgcgt caggcgacct
     3001 ctgaaattaa caaaaaattt ccagtcatcg aatttgattc tgtgcgatag cgcccctgtg
     3061 tgttctcgtt atgttgagga aaaaaataat ggttgctaag agattcgaac tcttgcatct
     3121 tacgatacct gagtattccc acagttaact gcggtcaaga tatttcttga atcaggcgcc
     3181 ttagaccgct cggccaaaca accaattact tgttgagaaa tagagtataa ttatcctata
     3241 aatataacgt ttttgaacac acatgaacaa ggaagtacag gacaattgat tttcgatcgg
     3301 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     3361 aacgacggcc agtgaattgt aatacgactc actatagggc gaattggaga aacagaataa
     3421 tatataaacc tgtataatat aaccttgaag actatatttc ttttcgcggc cgcctatagg
     3481 gcgaattgga gctccaccgc ggtggctcta gaactagtgg atcccccggg ctgcaggaat
     3541 tcgatatcaa gcttatcgat accgtcgacc tcgagggggg gcccggtacc cagcttcggc
     3601 cgtatttaca gataacgatg tcgttattat atatatatat atatattgtc acttgtaccc
     3661 agcttttgtt ccctttagtg agggttaatt tcgagcttgg cgtaatcatg gtcatagctg
     3721 tttcctgtgt gaaattgtta tccgctcaca acagctgtaa tatatagtag taacctgaaa
     3781 aatagtcaat gcccgtggtt catttgccaa tagaacagtc gatgtgatca atgtacacga
     3841 aagtcaatat aaaatatcca aatagccaat ggtgtcagga cggctaaggc aaatgcaact
     3901 ttgaaatact tattcggttt gcatcttggc gcgcctcttc ctgcaggcat taatgaatcg
     3961 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4021 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4081 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4141 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4201 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4261 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4321 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4381 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4441 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4501 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4561 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4621 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4681 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4741 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttgt ttaaacctac
     4801 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4861 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4921 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4981 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5041 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5101 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5161 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5221 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5281 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5341 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tggttgtcag
     5401 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5461 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5521 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5581 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5641 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5701 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5761 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5821 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5881 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacct
//
