LOCUS       sequence-160275-        7464 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector for integration at yeast Leu locus, expression 
            from the GPD promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7464)
  TITLE     Nemhauser plasmids
REFERENCE   2  (bases 1 to 7464)
  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"
COMMENT     Sequence Label: pGP5G-ccdB
FEATURES             Location/Qualifiers
     source          1..7464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2301..2319
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     terminator      complement(2356..2603)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     2569..2587
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     2605..2621
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2606..2622
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2655..2779
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(2820..3125)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(2922..2941)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(3470..4129)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4044..4063
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(4183..4189,4190..4207,4208..4213))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(4238..4362)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4370..4386)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4370..4386)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(4392..5035)
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     complement(4475..4498)
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     promoter        5294..5699
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5700..6794
                     /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
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(7295..7314)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7414..7436
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttt cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggca gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg ggggaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttac ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctc gggtttaaac acatcgagac caagaagaac attgctgatg
     2281 tgatgacaaa acctcttccg aattaaccct cactaaaggg aacaaaagct ggtaccgggc
     2341 cccccctcga cggccgcaaa ttaaagcctt cgagcgtccc aaaaccttct caagcaaggt
     2401 tttcagtata atgttacatg cgtacacgcg tctgtacaga aaaaaaagaa aaatttgaaa
     2461 tataaataac gttcttaata ctaacataac tataaaaaaa taaataggga cctagacttc
     2521 aggttgtcta actccttcct tttcggttag agcggatgtg gggggagggc gtgaatgtaa
     2581 gcgtgacata actaattaca tgactcgagg tcgacggtat cgataagctt gatatcgaat
     2641 tcctgcagcc catcaccact ttgtacaaga aagctgaacg agaaacgtaa aatgatataa
     2701 atatcaatat attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac
     2761 aacatatcca gtcactatgg tcgacctgca gactggctgt gtataaggga gcctgacatt
     2821 tatattcccc agaacatcag gttaatggcg tttttgatgt cattttcgcg gtggctgaga
     2881 tcagccactt cttccccgat aacggagacc ggcacactgg ccatatcggt ggtcatcatg
     2941 cgccagcttt catccccgat atgcaccacc gggtaaagtt cacgggagac tttatctgac
     3001 agcagacgtg cactggccag ggggatcacc atccgtcgcc cgggcgtgtc aataatatca
     3061 ctctgtacat ccacaaacag acgataacgg ctctctcttt tataggtgta aaccttaaac
     3121 tgcatttcac cagcccctgt tctcgtcagc aaaagagccg ttcatttcaa taaaccgggc
     3181 gacctcagcc atcccttcct gattttccgc tttccagcgt tcggcacgca gacgacgggc
     3241 ttcattctgc atggttgtgc ttaccagacc ggagatattg acatcatata tgccttgagc
     3301 aactgatagc tgtcgctgtc aactgtcact gtaatacgct gcttcatagc atacctcttt
     3361 ttgacatact tcgggtatac atatcagtat atattcttat accgcaaaaa tcagcgcgca
     3421 aatacgcata ctgttatctg gcttttagta agccggatcc acgcggcgtt tacgccccgc
     3481 cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat
     3541 cacagacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat
     3601 aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat
     3661 caaaactggt gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc
     3721 ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta
     3781 gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct
     3841 catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca
     3901 ttgccatacg gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg
     3961 gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa
     4021 cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat
     4081 gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct
     4141 tagctcctga aaatctcgac ggatcctaac tcaaaatcca cacattatac gagccggaag
     4201 cataaagtgt aaagcctggg gtgcctaatg cggccgccat agtgactgga tatgttgtgt
     4261 tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat
     4321 atcattttac gtttctcgtt cagctttttt gtacaaactt gtgatggggg atccactagt
     4381 tctagaatcc gtcgaaacta agttctggtg ttttaaaact aaaaaaaaga ctaactataa
     4441 aagtagaatt taagaagttt aagaaataga tttacagaat tacaatcaat acctaccgtc
     4501 tttatatact tattagtcaa gtaggggaat aatttcaggg aactggtttc aacctttttt
     4561 ttcagctttt tccaaatcag agagagcaga aggtaataga aggtgtaaga aaatgagata
     4621 gatacatgcg tgggtcaatt gccttgtgtc atcatttact ccaggcaggt tgcatcactc
     4681 cattgaggtt gtgcccgttt tttgcctgtt tgtgcccctg ttctctgtag ttgcgctaag
     4741 agaatggacc tatgaactga tggttggtga agaaaacaat attttggtgc tgggattctt
     4801 tttttttctg gatgccagct taaaaagcgg gctccattat atttagtgga tgccaggaat
     4861 aaactgttca cccagacacc tacgatgtta tatattctgt gtaacccgcc ccctattttg
     4921 ggcatgtacg ggttacagca gaattaaaag gctaattttt tgactaaata aagttaggaa
     4981 aatcactact attaattatt tacgtattct ttgaaatggc gagtattgat aatgataaac
     5041 tgcatatcga ccctcgagga gaacttctag tatatccaca tacctaatat tattgcctta
     5101 ttaaaaatgg aatcccaaca attacatcaa aatccacatt ctcttcaaaa tcaattgtcc
     5161 tgtacttcct tgttcatgtg tgttcaaaaa cgttatattt ataggataat tatactctat
     5221 ttctcaacaa gtaattggtt gtttggccga gcggtctaag gcgcctgatt caagaaatat
     5281 cttgaccgca gttaactgtg ggaatactca ggtatcgtaa gatgcaagag ttcgaatctc
     5341 ttagcaacca ttattttttt cctcaacata acgagaacac acaggggcgc tatcgcacag
     5401 aatcaaattc gatgactgga aattttttgt taatttcaga ggtcgcctga cgcatatacc
     5461 tttttcaact gaaaaattgg gagaaaaagg aaaggtgaga gcgccggaac cggcttttca
     5521 tatagaatag agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat
     5581 attatttaag gacctattgt tttttccaat aggtggttag caatcgtctt actttctaac
     5641 ttttcttacc ttttacattt cagcaatata tatatatatt tcaaggatat accattctaa
     5701 tgtctgcccc taagaagatc gtcgttttgc caggtgacca cgttggtcaa gaaatcacag
     5761 ccgaagccat taaggttctt aaagctattt ctgatgttcg ttccaatgtc aagttcgatt
     5821 tcgaaaatca tttaattggt ggtgctgcta tcgatgctac aggtgttcca cttccagatg
     5881 aggcgctgga agcctccaag aaggctgatg ccgttttgtt aggtgctgtg ggtggtccta
     5941 aatggggtac cggtagtgtt agacctgaac aaggtttact aaaaatccgt aaagaacttc
     6001 aattgtacgc caacttaaga ccatgtaact ttgcatccga ctctctttta gacttatctc
     6061 caatcaagcc acaatttgct aaaggtactg acttcgttgt tgtcagagaa ttagtgggag
     6121 gtatttactt tggtaagaga aaggaagacg atggtgatgg tgtcgcttgg gatagtgaac
     6181 aatacaccgt tccagaagtg caaagaatca caagaatggc cgctttcatg gccctacaac
     6241 atgagccacc attgcctatt tggtccttgg ataaagctaa tgttttggcc tcttcaagat
     6301 tatggagaaa aactgtggag gaaaccatca agaacgaatt ccctacattg aaggttcaac
     6361 atcaattgat tgattctgcc gccatgatcc tagttaagaa cccaacccac ctaaatggta
     6421 ttataatcac cagcaacatg tttggtgata tcatctccga tgaagcctcc gttatcccag
     6481 gttccttggg tttgttgcca tctgcgtcct tggcctcttt gccagacaag aacaccgcat
     6541 ttggtttgta cgaaccatgc cacggttctg ctccagattt gccaaagaat aaggttgacc
     6601 ctatcgccac tatcttgtct gctgcaatga tgttgaaatt gtcattgaac ttgcctgaag
     6661 aaggtaaggc cattgaagat gcagttaaaa aggttttgga tgcaggtatc agaactggtg
     6721 atttaggtgg ttccaacagt accaccgaag tcggtgatgc tgtcgccgaa gaagttaaga
     6781 aaatccttgc ttaaaaagat tctctttttt tatgatattt gtacataaac tttataaatg
     6841 aaattcataa tagaaacgac acgaaattac aaaatggaat atgttcatag ggtagacgaa
     6901 actatatacg caatctacat acatttatca agaaggagaa aaaggaggat gtaaaggaat
     6961 acaggtaagc aaattgatac taatggctca acgtgataag gaaaaagaat tgcactttaa
     7021 cattaatatt gacaaggagg agggcaccac acaaaaagtt aggtgtaaca gaaaatcatg
     7081 aaactatgat tcctaattta tatattggag gattttctct aaaaaaaaaa aaatacaaca
     7141 aataaaaaac actcaatgac ctgaccattt gatggagttt aagtcaatac cttcttgaac
     7201 catttcccat aatggtgaaa gttccctcaa gaattttact ctgtcagaaa cggccttacg
     7261 acgtagtcga gtttaaacta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     7321 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     7381 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     7441 caccgtcatc accgaaacgc gcga
//
