LOCUS       sequence-396554-        7087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast surface display destination vector for Golden Gate cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7087)
  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 7087)
  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: pND003
FEATURES             Location/Qualifiers
     source          1..7087
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     44..66
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        100..118
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        148..800
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    352..469
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     764..787
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             1119..1130
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1131..1157
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     RBS             1352..1363
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1370..2083
                     /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      2095..2166
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2182..2209
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             2224..2253
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2582..2601)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2583..2601)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2608..2625)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2608..2624)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2617..2639)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2765..3220
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2852..2871)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3062..3083
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3323..3997)
                     /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(3998..4278)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(4299..4318)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4418..4440
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4478..4496)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4537..5040
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5077..5181
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5182..5997
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5182..5997
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5255..5274)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6176..6764
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6665..6684
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6918..6935
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7052..7073
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgacc atgattacgc caagctcgaa attaaccctc actaaaggga
      121 acaaaagctg gtaccaattc cttgaatttt caaaaattct tacttttttt ttggatggac
      181 gcaaagaagt ttaataatca tattacatgg cattaccacc atatacatat ccatatctaa
      241 tcttacttat atgttgtgga aatgtaaaga gccccattat cttagcctaa aaaaaccttc
      301 tctttggaac tttcagtaat acgcttaact gctcattgct atattgaagt acggattaga
      361 agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt cctcgtcttc
      421 accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga acaataaaga
      481 ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac
      541 aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga ttagtttttt
      601 agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat
      661 ataaatgcaa aaactgcata accactttaa ctaatacttt caacattttc ggtttgtatt
      721 acttcttatt caaatgtaat aaaagtatca acaaaaaatt gttaatatac ctctatactt
      781 taacgtcaag gagaaaaaac cccggatcga attccctact tcatacattt tcaattaaga
      841 tgcagttact tcgctgtttt tcaatatttt ctgttattgc ttcagtttta gcacaggaac
      901 tgacaactat atgcgagcaa atcccctcac caactttaga atcgacgccg tactctttgt
      961 caacgactac tattttggcc aacgggaagg caatgcaagg agtttttgaa tattacaaat
     1021 cagtaacgtt tgtcagtaat tgcggttctc acccctcaac aactagcaaa ggcagcccca
     1081 taaacacaca gtatgttttt aaggacaata gctcgacgat tgaaggtaga tacccatacg
     1141 acgttccaga ctacgctctg caggctagtg gtggaggagg ctctggtgga ggcggttgag
     1201 accgaaagtg aaacgtgatt tcatgcgtca ttttgaacat tttgtaaatc ttatttaata
     1261 atgtgtgcgg caattcacat ttaatttatg aatgttttct taacatcgcg gcaactcaag
     1321 aaacggcagg ttcggatctt agctactaga gaaagaggag aaatactaga tgcgtaaagg
     1381 cgaagagctg ttcactggtg tcgtccctat tctggtggaa ctggatggtg atgtcaacgg
     1441 tcataagttt tccgtgcgtg gcgagggtga aggtgacgca actaatggta aactgacgct
     1501 gaagttcatc tgtactactg gtaaactgcc ggttccttgg ccgactctgg taacgacgct
     1561 gacttatggt gttcagtgct ttgctcgtta tccggaccat atgaagcagc atgacttctt
     1621 caagtccgcc atgccggaag gctatgtgca ggaacgcacg atttccttta aggatgacgg
     1681 cacgtacaaa acgcgtgcgg aagtgaaatt tgaaggcgat accctggtaa accgcattga
     1741 gctgaaaggc attgacttta aagaggacgg caatatcctg ggccataagc tggaatacaa
     1801 ttttaacagc cacaatgttt acatcaccgc cgataaacaa aaaaatggca ttaaagcgaa
     1861 ttttaaaatt cgccacaacg tggaggatgg cagcgtgcag ctggctgatc actaccagca
     1921 aaacactcca atcggtgatg gtcctgttct gctgccagac aatcactatc tgagcacgca
     1981 aagcgttctg tctaaagatc cgaacgagaa acgcgatcat atggttctgc tggagttcgt
     2041 aaccgcagcg ggcatcacgc atggtatgga tgaactgtac aaatgaccag gcatcaaata
     2101 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     2161 gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttataggtct
     2221 cacgaacaaa agcttatttc tgaagaggac ttgtaataga gatctgataa caacagtgta
     2281 gatgtaacaa aatcgacttt gttcccactg tacttttagc tcgtacaaaa tacaatatac
     2341 ttttcatttc tccgtaaaca acatgttttc ccatgtaata tccttttcta tttttcgttc
     2401 cgttaccaac tttacacata ctttatatag ctattcactt ctatacacta aaaaactaag
     2461 acaattttaa ttttgctgcc tgccatattt caatttgtta taaattccta taatttatcc
     2521 tattagtagc taaaaaaaga tgaatgtgaa tcgaatccta agagaattga gctccaattc
     2581 gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga
     2641 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     2701 taatagcgaa gaggcccgca ccgatcgcct ttcccaacag ttgcgcagcc tgaatggcga
     2761 atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2821 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2881 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2941 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     3001 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3061 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     3121 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     3181 tttaacgcga attttaacaa aatattaacg cttacaattt cctgatgcgg tattttctcc
     3241 ttacgcatct gtgcggtatt tcacaccgca tagatcggca agtgcacaaa caatacttaa
     3301 ataaatacta ctcagtaata acctatttct tagcattttt gacgaaattt gctattttgt
     3361 tagagtcttt tacaccattt gtctccacac ctccgcttac atcaacacca ataacgccat
     3421 ttaatctaag cgcatcacca acattttctg gcgtcagtcc accagctaac ataaaatgta
     3481 agctttcggg gctctcttgc cttccaaccc agtcagaaat cgagttccaa tccaaaagtt
     3541 cacctgtccc acctgcttct gaatcaaaca agggaataaa cgaatgaggt ttctgtgaag
     3601 ctgcactgag tagtatgttg cagtcttttg gaaatacgag tcttttaata actggcaaac
     3661 cgaggaactc ttggtattct tgccacgact catctccatg cagttggacg atatcaatgc
     3721 cgtaatcatt gaccagagcc aaaacatcct ccttaggttg attacgaaac acgccaacca
     3781 agtatttcgg agtgcctgaa ctatttttat atgcttttac aagacttgaa attttccttg
     3841 caataaccgg gtcaattgtt ctctttctat tgggcacaca tataataccc agcaagtcag
     3901 catcggaatc tagagcacat tctgcggcct ctgtgctctg caagccgcaa actttcacca
     3961 atggaccaga actacctgtg aaattaataa cagacatact ccaagctgcc tttgtgtgct
     4021 taatcacgta tactcacgtg ctcaatagtc accaatgccc tccctcttgg ccctctcctt
     4081 ttcttttttc gaccgaatta attcttaatc ggcaaaaaaa gaaaagctcc ggatcaagat
     4141 tgtacgtaag gtgacaagct atttttcaat aaagaatatc ttccactact gccatctggc
     4201 gtcataactg caaagtacac atatattacg atgctgttct attaaatgct tcctatatta
     4261 tatatatagt aatgtcgtga tctatggtgc actctcagta caatctgctc tgatgccgca
     4321 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4381 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4441 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     4501 taggttaatg tcatgataat aatggtttct tagacggatc gcttgcctgt aacttacacg
     4561 cgcctcgtat cttttaatga tggaataatt tgggaattta ctctgtgttt atttattttt
     4621 atgttttgta tttggatttt agaaagtaaa taaagaaggt agaagagtta cggaatgaag
     4681 aaaaaaaaat aaacaaaggt ttaaaaaatt tcaacaaaaa gcgtacttta catatatatt
     4741 tattagacaa gaaaagcaga ttaaatagat atacattcga ttaacgataa gtaaaatgta
     4801 aaatcacagg attttcgtgt gtggtcttct acacagacaa gatgaaacaa ttcggcatta
     4861 atacctgaga gcaggaagag caagataaaa ggtagtattt gttggcgatc cccctagagt
     4921 cttttacatc ttcggaaaac aaaaactatt ttttctttaa tttctttttt tactttctat
     4981 ttttaattta tatatttata ttaaaaaatt taaattataa ttatttttat agcacgtgat
     5041 gaaaaggacc caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5101 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5161 taatattgaa aaaggaagag tatgagccat attcaacggg aaacgtcttg ctctaggccg
     5221 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
     5281 gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt
     5341 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     5401 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     5461 gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat
     5521 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     5581 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa
     5641 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     5701 cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc
     5761 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     5821 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     5881 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     5941 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaacct
     6001 cagcaactgt caaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6061 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6121 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6181 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6241 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6301 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6361 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6421 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6481 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6541 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6601 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6661 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6721 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6781 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6841 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     6901 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     6961 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     7021 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     7081 cccaggc
//
