LOCUS       sequence-430589-        7262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast surface display Fab vector with kappa light chain and mRFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7262)
  AUTHORS   Petersen BM, Kirby MB, Chrispens KM, Irvin OM, Strawn IK, Haas CM, 
            Walker AM, Baumer ZT, Ulmer SA, Ayala E, Rhodes ER, Guthmiller JJ, 
            Steiner PJ, Whitehead TA
  TITLE     An integrated technology for quantitative wide mutational scanning 
            of human antibody Fab libraries.
  JOURNAL   Nat Commun. 2024 May 10;15(1):3974. doi: 10.1038/s41467-024-48072-z.
  PUBMED    38730230
REFERENCE   2  (bases 1 to 7262)
  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: "Nat 
            Commun."; date: "2024-05-10"; volume: "15(1):3974. doi"; pages: " 
            10.1038/s41467-024-48072-z"
COMMENT     Sequence Label: pYSD_kappa_mRFP
FEATURES             Location/Qualifiers
     source          1..7262
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    2..18
                     /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     7..29
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     26..42
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     26..42
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        63..81
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     terminator      complement(123..288)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     209..233
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(442..483)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1442..1471
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1800..1819)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1801..1819)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1826..1843)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1826..1842)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1835..1857)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    1983..2001
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2008..2026
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             2044..2055
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2062..2739
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      2757..2828
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2844..2871
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      2903..3358
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2990..3009)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3200..3221
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3461..4135)
                     /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(4136..4416)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(4437..4456)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4556..4578
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4616..4634)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4675..5178
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5215..5319
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5320..6135
                     /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             5320..6135
                     /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(5393..5412)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6314..6902
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6803..6822
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7056..7073
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7190..7211
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7226..7231,7232..7249,7250..7256)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc
       61 gaaattaacc ctcactaaag ggaacaaaag ctggtacctt cgctattacg ccagctgaat
      121 tggagcgacc tcatgctata cctgagaaag caacctgacc tacaggaaag agttactcaa
      181 gaataagaat tttcgtttta aaacctaaga gtcactttaa aatttgtata cacttatttt
      241 ttttataact tatttaataa taaaaatcat aaatcataag aaattcgctt atttagaagt
      301 gtcaacaacg tatctaccaa cgatttgacc cttttccatc ttttcgtaaa tttctggcaa
      361 ggtagacaag ccgacaacct tgattggaga cttgaccaaa cctctggcga agaattgtta
      421 attaagagct cagatctatt aggtggaatc caggcccagc agcgggttcg gaatcggttt
      481 gccacattca cccctgttga aagacttagt aactggagaa gataaacctt ggtgggtaac
      541 ttcacaagcg taaaccttat gtttttcgta atcggccttg gacaaagtca aagtagatga
      601 caaagagtag gtagagtcct tggaatcttg ttcagtaaca gattcttggg agttaccaga
      661 ctgcaatgcg ttatcaactt tccattgaac cttagcttct cttgggtaga aattgttcaa
      721 cagacaaaca acagaagcag taccagactt caactgttca tcagatggtg gaaaaatgaa
      781 gacggatgga gcagctacag gcggccgcag ctacacttca ttggatggat gaatcgcggc
      841 cgccaagggc ccgcaggtgt tccccctggc ccccagcagc aagagcacca gcggcggcac
      901 agccgccctg ggctgcctgg tgaaggacta cttccccgag cccgtgaccg tgagctggaa
      961 cagcggcgcc ttgaccagcg gcgtgcacac cttccccgcc gtgctgcaga gcagcggcct
     1021 gtacagcctg agcagcgtgg tgaccgtgcc cagcagcagc ctgggcaccc agacctacat
     1081 ctgcaacgtg aaccacaagc ccagcaacac caaggtggac aaacgcgtgg agcccaagag
     1141 ctgcgataag actcataccg gtggtggtgg ttctggtggt ggtggttctg gtggtggtgg
     1201 ttctggtggt ggtggttctc aggaactgac aactatatgc gagcaaatcc cctcaccaac
     1261 tttagaatcg acgccgtact ctttgtcaac gactactatt ttggccaacg ggaaggcaat
     1321 gcaaggagtt tttgaatatt acaaatcagt aacgtttgtc agtaattgcg gttctcaccc
     1381 ctcaacaact agcaaaggca gccccataaa cacacagtat gtttttggtg ggggcggatc
     1441 cgaacaaaag cttatttctg aagaggactt gtaatagaga tctgataaca acagtgtaga
     1501 tgtaacaaaa tcgactttgt tcccactgta cttttagctc gtacaaaata caatatactt
     1561 ttcatttctc cgtaaacaac atgttttccc atgtaatatc cttttctatt tttcgttccg
     1621 ttaccaactt tacacatact ttatatagct attcacttct atacactaaa aaactaagac
     1681 aattttaatt ttgctgcctg ccatatttca atttgttata aattcctata atttatccta
     1741 ttagtagcta aaaaaagatg aatgtgaatc gaatcctaag agaattgagc tccaattcgc
     1801 cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     1861 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     1921 atagcgaaga ggcccgcacc gatcgccttt cccaacagtt gcgcagcctt gcctgagacc
     1981 agtccctatc agtgatagag attgacatcc ctatcagtga tagagatact gagcacggat
     2041 ctgaaagagg agaaaggatc tatggcgagt agcgaagacg ttatcaaaga gttcatgcgt
     2101 ttcaaagttc gtatggaagg ttccgttaac ggtcacgagt tcgaaatcga aggtgaaggt
     2161 gaaggtcgtc cgtacgaagg tactcagacc gctaaactga aagttaccaa aggtggtccg
     2221 ctgccgttcg cttgggacat cctgtccccg cagttccagt acggttccaa agcttacgtt
     2281 aaacacccgg ctgacatccc ggactacctg aaactgtcct tcccggaagg tttcaaatgg
     2341 gaacgtgtta tgaacttcga agacggtggt gttgttaccg ttacccagga ctcctccctg
     2401 caagacggtg agttcatcta caaagttaaa ctgcgtggta ctaacttccc gtccgacggt
     2461 ccggttatgc agaaaaaaac catgggttgg gaagcttcca ccgaacgtat gtacccggaa
     2521 gacggtgctc tgaaaggtga aatcaaaatg cgtctgaaac tgaaagacgg tggtcactac
     2581 gacgctgaag ttaaaaccac ctacatggct aaaaaaccgg ttcagctgcc gggtgcttac
     2641 aaaaccgaca tcaaactgga catcacctcc cacaacgaag actacaccat cgttgaacag
     2701 tacgaacgtg ctgaaggtcg tcactccacc ggtgcttaat aaggatctcc aggcatcaaa
     2761 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
     2821 acgctctcta ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttataagt
     2881 cggtctcaac tatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2941 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3001 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3061 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3121 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3181 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3241 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3301 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc
     3361 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata gatcggcaag
     3421 tgcacaaaca atacttaaat aaatactact cagtaataac ctatttctta gcatttttga
     3481 cgaaatttgc tattttgtta gagtctttta caccatttgt ctccacacct ccgcttacat
     3541 caacaccaat aacgccattt aatctaagcg catcaccaac attttctggc gtcagtccac
     3601 cagctaacat aaaatgtaag ctttcggggc tctcttgcct tccaacccag tcagaaatcg
     3661 agttccaatc caaaagttca cctgtccctc ctgcttctga atcaaacaag ggaataaacg
     3721 aatgaggttt ctgtgaagct gcactgagta gtatgttgca gtcttttgga aatacgagtc
     3781 ttttaataac tggcaaaccg aggaactctt ggtattcttg ccacgactca tctccatgca
     3841 gttggacgat atcaatgccg taatcattga ccagagccaa aacatcctcc ttaggttgat
     3901 tacgaaacac gccaaccaag tatttcggag tgcctgaact atttttatat gcttttacaa
     3961 gacttgaaat tttccttgca ataaccgggt caattgttct ctttctattg ggcacacata
     4021 taatacccag caagtcagca tcggaatcta gagcacattc tgcggcctct gtgctctgca
     4081 agccgcaaac tttcaccaat ggaccagaac tacctgtgaa attaataaca gacatactcc
     4141 aagctgcctt tgtgtgctta atcacgtata ctcacgtgct caatagtcac caatgccctc
     4201 cctcttggcc ctctcctttt cttttttcga ccgaattaat tcttaatcgg caaaaaaaga
     4261 aaagctccgg atcaagattg tacgtaaggt gacaagctat ttttcaataa agaatatctt
     4321 ccactactgc catctggcgt cataactgca aagtacacat atattacgat gctgttctat
     4381 taaatgcttc ctatattata tatatagtaa tgtcgtgatc tatggtgcac tctcagtaca
     4441 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     4501 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4561 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     4621 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacggatcgc
     4681 ttgcctgtaa cttacacgcg cctcgtatct tttaatgatg gaataatttg ggaatttact
     4741 ctgtgtttat ttatttttat gttttgtatt tggattttag aaagtaaata aagaaggtag
     4801 aagagttacg gaatgaagaa aaaaaaataa acaaaggttt aaaaaatttc aacaaaaagc
     4861 gtactttaca tatatattta ttagacaaga aaagcagatt aaatagatat acattcgatt
     4921 aacgataagt aaaatgtaaa atcacaggat tttcgtgtgt ggtcgtctac acagacaaga
     4981 tgaaacaatt cggcattaat acctgagagc aggaagagca agataaaagg tagtatttgt
     5041 tggcgatccc cctagagtct tttacatctt cggaaaacaa aaactatttt ttctttaatt
     5101 tcttttttta ctttctattt ttaatttata tatttatatt aaaaaattta aattataatt
     5161 atttttatag cacgtgatga aaaggaccca ggtggcactt ttcggggaaa tgtgcgcgga
     5221 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5281 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagccatat tcaacgggaa
     5341 acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     5401 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
     5461 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     5521 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     5581 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     5641 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     5701 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     5761 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     5821 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     5881 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     5941 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     6001 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     6061 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     6121 gatgagtttt tctaacctca gcaactgtca accaagttta ctcatatata ctttagattg
     6181 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6241 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6301 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6361 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6421 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6481 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6541 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6601 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6661 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6721 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6781 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6841 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6901 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6961 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     7021 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     7081 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     7141 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     7201 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     7261 ga
//
