LOCUS       62056_18105        7105 bp DNA     circular SYN 07-JAN-2021
DEFINITION  Yeast display vector pNMB1m_C_Nb207, complete sequence.
ACCESSION   MT543226
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7105)
  AUTHORS   Uchanski T, Masiulis S, Fischer B, Kalichuk V, Lopez-Sanchez U, 
            Zarkadas E, Weckener M, Sente A, Ward P, Wohlkoenig A, Zoegg T, 
            Remaut H, Naismith JH, Nury H, Vranken W, Aricescu AR, Pardon E, 
            Steyaert J.
  TITLE     Megabodies expand the nanobody toolkit for protein structure 
            determination by single-particle cryo-EM
  JOURNAL   Nat Methods 18 (1), 60-68 (2021)
REFERENCE   2  (bases 1 to 7105)
  AUTHORS   Steyaert J, Uchanski T, Pardon E.
  TITLE     Direct Submission
  JOURNAL   Submitted (31-MAY-2020) VIB-VUB Center for Structural Biology, VIB, 
            Pleinlaan 2, Brussels 1050, Belgium
REFERENCE   3  (bases 1 to 7105)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods"; date: "2021"; volume: "18"; issue: "1"; pages: "60-68"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (31-MAY-2020) VIB-VUB Center for Structural Biology, VIB, Pleinlaan 
            2, Brussels 1050, Belgium"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7105
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    complement(70..573)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
     promoter        610..714
                     /label=AmpR promoter
     CDS             715..1572
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1746..2334
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2622..2643
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2658..2688
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2696..2712
                     /label=lac operator
                     /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     2720..2736
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2757..2775
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2940..3592
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     misc_feature    3632..3898
                     /label=appS4 leader sequence
                     /note="appS4 leader sequence"
     misc_feature    3899..3934
                     /label=Nanobody beta-strain A (residues 1-12)
                     /note="Nanobody beta-strain A (residues 1-12)"
     misc_feature    3932..3964
                     /label=multi cloning site
                     /note="multi cloning site"
     misc_feature    3962..4291
                     /label=Nanobody Nb207 C-terminal part (residues 18-128)
                     /note="Nanobody Nb207 C-terminal part (residues 18-128)"
     misc_feature    4295..4396
                     /label=linker
                     /note="linker"
     misc_feature    4397..4603
                     /label=Aga2p anchor protein
                     /note="Aga2p anchor protein"
     CDS             4625..4858
                     /label=ACP
                     /note="acyl carrier protein (ACP) from E. coli"
     misc_feature    4862..4891
                     /label=Myc-tag
                     /note="Myc-tag"
     regulatory      4905..5211
                     /label=MFa1
                     /note="MFa1"
                     /regulatory_class="terminator"
     promoter        complement(5226..5244)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5251..5267)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      5412..5867
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(5967..6638)
                     /label=TRP1
                     /note="phosphoribosylanthranilate isomerase, required for 
                     tryptophan biosynthesis"
     promoter        complement(6639..6919)
                     /label=TRP1 promoter
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata
      121 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt
      181 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa
      241 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata
      301 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct
      361 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gggcaagata
      421 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact
      481 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa
      541 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg
      601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
      961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1141 ccgaaggagc taaccgcttt ttttcacaac atgggggatc atgtaactcg ccttgatcgt
     1201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1501 ggcagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1861 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     1921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2101 acgacctaca ccgaactgag atacctacag cgtgagcatt gagaaagcgc cacgcttccc
     2161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2221 agggagcttc caggggggaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc cgagcctatg gaaaaacgcc
     2341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagggcgc
     2521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact
     2641 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg
     2701 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcggaatt
     2761 aaccctcact aaagggaaca aaagctgggt acccgacagg ttatcagcaa caacacagtc
     2821 atatccattc tcaattagct ctaccacagt gtgtgaacca atgtatccag caccacctgt
     2881 aaccaaaaca attttagaag tactttcact ttgtaactga gctgtcattt atattgaatt
     2941 ttcaaaaatt cttacttttt ttttggatgg acgcaaagaa gtttaataat catattacat
     3001 ggcattacca ccatatacat atccatatct aatcttactt atatgttgtg gaaatgtaaa
     3061 gagccccatt atcttagcct aaaaaaacct tctctttgga actttcagta atacgcttaa
     3121 ctgctcattg ctatattgaa gtacggatta gaagccgccg agcgggtgac agccctccga
     3181 aggaagactc tcctccgtgc gtcctcgtct tcaccggtcg cgttcctgaa acgcagatgt
     3241 gcctcgcgcc gcactgctcc gaacaataaa gattctacaa tactagcttt tatggttatg
     3301 aagaggaaaa attggcagta acctggcccc acaaaccttc aaatgaacga atcaaattaa
     3361 caaccatagg atgataatgc gattagtttt ttagccttat ttctggggta attaatcagc
     3421 gaagcgatga tttttgatct attaacagat atataaatgc aaaaactgca taaccacttt
     3481 aactaatact ttcaacattt tcggtttgta ttacttctta ttcaaatgta ataaaagtat
     3541 caacaaaaaa ttgttaatat acctctatac tttaacgtca aggagaaaaa accccggatc
     3601 gaattcccta cttcatacat tttcaattaa gatgagattt ccttcaattt ttactgctgt
     3661 tgtcttcgca gcatcctccg cattagctgc tccagcaaac actacagctg aagatgaaac
     3721 ggcacaaatt ccagctgaag ctgtcatcgg ttacttaggt ttagaagggg atagcgatgt
     3781 tgctgccttg ccattatccg atagcacaaa taacgggtca ttgtccacaa atactactat
     3841 tgccagcatt gctgctaaag aagaaggggt acaattagat aaaagagagg ccgaagcaca
     3901 ggtgcagctg gtggagtctg ggggaggact agtaggaaga gcttagtcga caagctcttc
     3961 atccctgaga ctctcctgtg cagcctctgg acgcaccttc agtaccgctg ccatgggctg
     4021 gttccgccag gctccaggaa aggagcgtga cttcgtagca ggtatttact ggactgttgg
     4081 cagcacatac tatgcagact ccgcgaaggg ccgattcacc atctccagag acaacgccaa
     4141 gaacacggtg tatctgcaaa tggacagcct gaaaccagag gacacggccg tttattactg
     4201 tgcagctagg cggaggggct ttactttagc ccctactcga gcaaatgaat atgactactg
     4261 gggtcagggg acccaggtca ccgtctcaag cttgggaggt ggctctggtg gagggggatc
     4321 aggaggtggt ggctccggtg gaggtggttc gggcggaggt ggttccggtg gaggaggttc
     4381 tggtggaggc ggtagccagg aactgacaac tatatgcgag caaatcccct caccaacttt
     4441 agaatcgaca ccatactctt tgtcaacgac tactatttta gccaacggga aggcaatgca
     4501 aggagttttt gaatattaca aatcagtaac gtttgtcagt aattgcggtt ctcacccctc
     4561 aacaactagc aaaggcagcc ccataaacac acagtatgtt tttaaggaca atagctcgac
     4621 aagtatgagc actatcgaag aacgtgttaa gaaaattatc ggcgaacagc tgggcgttaa
     4681 gcaggaagaa gttaccaaca atgcttcttt cgttgaagac ctgggcgccg attctcttga
     4741 caccgttgag ctggtaatgg ctctggaaga agagtttgat actgagattc cagacgaaga
     4801 agctgagaaa atcaccaccg ttcaggctgc cattgattac atcaacggcc accaggcaag
     4861 cgaacaaaag ttaatttctg aagaggactt gtaatagcat gcagatctga taacaacagt
     4921 gtagatgtaa caaaatcgac tttgttccca ctgtactttt agctcgtaca aaatacaata
     4981 tacttttcat ttctccgtaa acaacatgtt ttcccatgta atatcctttt ctatttttcg
     5041 ttccgttacc aactttacac atactttata tagctattca cttctataca ctaaaaaact
     5101 aagacaattt taattttgct gcctgccata tttcaatttg ttataaattc ctataattta
     5161 tcctattagt agctaaaaaa agatgaatgt gaatcgaatc ctaagagaat tgagctccaa
     5221 ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg
     5281 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catcccccct tcgccagctg
     5341 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     5401 cgaatggcgc gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     5461 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     5521 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     5581 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     5641 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     5701 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     5761 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     5821 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcct gatgcggtat
     5881 tttctcctta cgcatctgtg cggtatttca caccgcaggc aagtgcacaa acaatactta
     5941 aataaatact actcagtaat aacctatttc ttagcatttt tgacgaaatt tgctattttg
     6001 ttagagtctt ttacaccatt tgtctccaca cctccgctta catcaacacc aataacgcca
     6061 tttaatctaa gcgcatcacc aacattttct ggcgtcagtc caccagctaa cataaaatgt
     6121 aaactttcgg ggctctcttg ccttccaacc cagtcagaaa tcgagttcca atccaaaagt
     6181 tcacctgtcc cacctgcttc tgaatcaaac aagggaataa acgaatgagg tttctgtgaa
     6241 gctgcactga gtagtatgtt gcagtctttt ggaaatacga gtcttttaat aactggcaaa
     6301 ccgaggaact cttggtattc ttgccacgac tcatctccat gcagttggac gatatcaatg
     6361 ccgtaatcat tgaccagagc caaaacatcc tccttaggtt gattacgaaa cacgccaacc
     6421 aagtatttcg gagtgcctga actattttta tatgctttta caagacttga aattttcctt
     6481 gcaataaccg ggtcaattgt tctctttcta ttgggcacac atataatacc cagcaagtca
     6541 gcatcggaat ctagagcaca ttctgcggcc tctgtgctct gcaagccgca aactttcacc
     6601 aatggaccag aactacctgt gaaattaata acagacatac tccaagctgc ctttgtgtgc
     6661 ttaatcacgt atactcacgt gctcaatagt caccaatgcc ctccctcttg gccctctcct
     6721 tttctttttt cgaccgaatt aattcttaat cggcaaaaaa agaaaagctc cggatcaaga
     6781 ttgtacgtaa ggtgacaagc tatttttcaa taaagaatat cttccactac tgccatctgg
     6841 cgtcataact gcaaagtaca catatattac gatgctgtct attaaatgct tcctatatta
     6901 tatatatagt aatgtcgttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     6961 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     7021 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     7081 tcaccgtcat caccgaaacg cgcga
//
