LOCUS       sequence-396569-        6996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast two-hybrid destination vector for Golden Gate cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6996)
  AUTHORS   Daffern N, Francino-Urdaniz IM, Baumer ZT, Whitehead TA
  TITLE     Standardizing cassette-based deep mutagenesis by Golden Gate 
            assembly.
  JOURNAL   Biotechnol Bioeng. 2024 Jan;121(1):281-290. doi: 10.1002/bit.28564. 
            Epub 2023 Sep 26.
  PUBMED    37750676
REFERENCE   2  (bases 1 to 6996)
  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"; doi: "10.1002/bit.28564";
            journalName: "Biotechnol Bioeng"; date: "2024-01"; volume: "121"; 
            issue: "1"; pages: "281-290"
COMMENT     Sequence Label: pND005
FEATURES             Location/Qualifiers
     source          1..6996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..406
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     RBS             584..595
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             602..1315
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1327..1398
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1414..1441
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      1462..1649
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1517..1541)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(1702..2376)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(2377..2478)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(2492..2508)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2492..2508)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2505..2527)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2516..2532
                     /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(2540..2546,2547..2564,2565..2570))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2585..2606
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        complement(2678..2782)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2809..3264
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2896..2915)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3106..3127
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3359..3376)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3530..4118)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3610..3629)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4575..4677
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4678..5337
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4744..4763)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     5618..5636
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5674..5696)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5796..5815
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      5832..6996
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 gcttgcatgc aacttctttt cttttttttt cttttctctc tcccccgttg ttgtctcacc
       61 atatccgcaa tgacaaaaaa atgatggaag acactaaagg aaaaaattaa cgacaaagac
      121 agcaccaaca gatgtcgttg ttccagagct gatgaggggt atctcgaagc acacgaaact
      181 ttttccttcc ttcattcacg cacactactc tctaatgagc aacggtatac ggccttcctt
      241 ccagttactt gaatttgaaa taaaaaaaag tttgctgtct tgctatcaag tataaataga
      301 cctgcaatta ttaatctttt gtttcctcgt cattgttctc gttccctttc ttccttgttt
      361 ctttttctgc acaatatttc aagctatacc aagcatacaa tcaactccaa gcttgaagca
      421 agcctccttg agaccgaaag tgaaacgtga tttcatgcgt cattttgaac attttgtaaa
      481 tcttatttaa taatgtgtgc ggcaattcac atttaattta tgaatgtttt cttaacatcg
      541 cggcaactca agaaacggca ggttcggatc ttagctacta gagaaagagg agaaatacta
      601 gatgcgtaaa ggcgaagagc tgttcactgg tgtcgtccct attctggtgg aactggatgg
      661 tgatgtcaac ggtcataagt tttccgtgcg tggcgagggt gaaggtgacg caactaatgg
      721 taaactgacg ctgaagttca tctgtactac tggtaaactg ccggttcctt ggccgactct
      781 ggtaacgacg ctgacttatg gtgttcagtg ctttgctcgt tatccggacc atatgaagca
      841 gcatgacttc ttcaagtccg ccatgccgga aggctatgtg caggaacgca cgatttcctt
      901 taaggatgac ggcacgtaca aaacgcgtgc ggaagtgaaa tttgaaggcg ataccctggt
      961 aaaccgcatt gagctgaaag gcattgactt taaagaggac ggcaatatcc tgggccataa
     1021 gctggaatac aattttaaca gccacaatgt ttacatcacc gccgataaac aaaaaaatgg
     1081 cattaaagcg aattttaaaa ttcgccacaa cgtggaggat ggcagcgtgc agctggctga
     1141 tcactaccag caaaacactc caatcggtga tggtcctgtt ctgctgccag acaatcacta
     1201 tctgagcacg caaagcgttc tgtctaaaga tccgaacgag aaacgcgatc atatggttct
     1261 gctggagttc gtaaccgcag cgggcatcac gcatggtatg gatgaactgt acaaatgacc
     1321 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     1381 ttgtcggtga acgctctcta ctagagtcac actggctcac cttcgggtgg gcctttctgc
     1441 gtttataggt ctcaattccg ggcgaatttc ttatgattta tgatttttat tattaaataa
     1501 gttataaaaa aaataagtgt atacaaattt taaagtgact cttaggtttt aaaacgaaaa
     1561 ttcttattct tgagtaactc tttcctgtag gtcaggttgc tttctcaggt atagcatgag
     1621 gtcgctctta ttgaccacac ctctaccggc atgccggcaa gtgcacaaac aatacttaaa
     1681 taaatactac tcagtaataa cctatttctt agcatttttg acgaaatttg ctattttgtt
     1741 agagtctttt acaccatttg tctccacacc tccgcttaca tcaacaccaa taacgccatt
     1801 taatctaagc gcatcaccaa cattttctgg cgtcagtcca ccagctaaca taaaatgtaa
     1861 gctttcgggg ctctcttgcc ttccaaccca gtcagaaatc gagttccaat ccaaaagttc
     1921 acctgtccca cctgcttctg aatcaaacaa gggaataaac gaatgaggtt tctgtgaagc
     1981 tgcactgagt agtatgttgc agtcttttgg aaatacgagt cttttaataa ctggcaaacc
     2041 gaggaactct tggtattctt gccacgactc atctccatgc agttggacga tatcaatgcc
     2101 gtaatcattg accagagcca aaacatcctc cttaggttga ttacgaaaca cgccaaccaa
     2161 gtatttcgga gtgcctgaac tatttttata tgcttttaca agacttgaaa ttttccttgc
     2221 aataaccggg tcaattgttc tctttctatt gggcacacat ataataccca gcaagtcagc
     2281 atcggaatct agagcacatt ctgcggcctc tgtgctctgc aagccgcaaa ctttcaccaa
     2341 tggaccagaa ctacctgtga aattaataac agacatactc caagctgcct ttgtgtgctt
     2401 aatcacgtat actcacgtgc tcaatagtca ccaatgccct ccctcttggc cctctccttt
     2461 tcttttttcg accgaattaa ttcgtaatca tgtcatagct gtttcctgtg tgaaattgtt
     2521 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     2581 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     2641 gaaacctgtc gtgccagctg gttctttccg cctcaggact cttccttttt caatattatt
     2701 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     2761 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gcgccctgta
     2821 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2881 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2941 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3001 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3061 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3121 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3181 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     3241 acaaaatatt aacgcttaca atttacgcgc ctaaattatg cggccgcata taactgcatt
     3301 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3361 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3421 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3481 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3541 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3601 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3661 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3721 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3781 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3841 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3901 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3961 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4021 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4081 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4141 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4201 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4261 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4321 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4381 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gatccacgct
     4441 caccggctcc agatttatca gcaataaacc agccagccgc ctgtgacgga agatcacttc
     4501 gcagaataaa taaatcctgg tgtccctgtt gataccggga agccctgggc caacttttgg
     4561 cgaaaatgag acgttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga
     4621 tcactaccgg gcgtattttt tgagttgtcg agattttcag gagctaagga agctaaaatg
     4681 gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat
     4741 tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt
     4801 acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac
     4861 attcttgccc gcctgatgaa tgctcatccg gaattacgta tggcaatgaa agacggtgag
     4921 ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg
     4981 ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca catatattcg
     5041 caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat
     5101 atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc
     5161 aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac
     5221 aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc
     5281 ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt
     5341 ttttaaggca gttattggtg cccttaaacg cctggttgct acgcctgaat aagtgataat
     5401 aagcggatga atggcagaaa ttcgaaagca aattcgaccc ggtcgtcggt tcagggcagg
     5461 gtcgttaaat agccgcttat gtctattgct ggtttaccgg tttattgact accggaagca
     5521 gtgtgaccgt gtgcttctca aatgcctgag gccagtttgc tcaggctctc cccgtggagg
     5581 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     5641 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     5701 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     5761 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     5821 agtgcaccat aacgcattta agcataaaca cgcactatgc cgttcttctc atgtatatat
     5881 atatacaggc aacacgcaga tataggtgcg acgtgaacag tgagctgtat gtgcgcagct
     5941 cgcgttgcat tttcggaagc gctcgttttc ggaaacgctt tgaagttcct attccgaagt
     6001 tcctattctc tagctagaaa gtataggaac ttcagagcgc ttttgaaaac caaaagcgct
     6061 ctgaagacgc actttcaaaa aaccaaaaac gcaccggact gtaacgagct actaaaatat
     6121 tgcgaatacc gcttccacaa acattgctca aaagtatctc tttgctatat atctctgtgc
     6181 tatatcccta tataacctac ccatccacct ttcgctcctt gaacttgcat ctaaactcga
     6241 cctctacatt ttttatgttt atctctagta ttactcttta gacaaaaaaa ttgtagtaag
     6301 aactattcat agagtgaatc gaaaacaata cgaaaatgta aacatttcct atacgtagta
     6361 tatagagaca aaatagaaga aaccgttcat aattttctga ccaatgaaga atcatcaacg
     6421 ctatcacttt ctgttcacaa agtatgcgca atccacatcg gtatagaata taatcgggga
     6481 tgcctttatc ttgaaaaaat gcacccgcag cttcgctagt aatcagtaaa cgcgggaagt
     6541 ggagtcaggc tttttttatg gaagagaaaa tagacaccaa agtagccttc ttctaacctt
     6601 aacggaccta cagtgcaaaa agttatcaag agactgcatt atagagcgca caaaggagaa
     6661 aaaaagtaat ctaagatgct ttgttagaaa aatagcgctc tcgggatgca tttttgtaga
     6721 acaaaaaaga agtatagatt ctttgttggt aaaatagcgc tctcgcgttg catttctgtt
     6781 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
     6841 gttttacaaa aatgaagcac agattcttcg ttggtaaaat agcgctttcg cgttgcattt
     6901 ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca
     6961 tttttgttct acaaaatgaa gcacagatgc ttcgtt
//
