LOCUS       62056_18250        8218 bp DNA     circular SYN 07-JAN-2021
DEFINITION  Yeast display vector pNS1MB, complete sequence.
ACCESSION   MT543227
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8218)
  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 8218)
  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 8218)
  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"
FEATURES             Location/Qualifiers
     source          1..8218
                     /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..3937
                     /label=NS1 monobody beta-strain A (residues 4-16)
                     /note="NS1 monobody beta-strain A (residues 4-16)"
     misc_feature    3938..3940
                     /label=Phe residue
                     /note="Phe residue"
     misc_feature    3941..5161
                     /label=cHopQ scaffold protein
                     /note="cHopQ scaffold protein"
     misc_feature    5162..5164
                     /label=Gly residue
                     /note="Gly residue"
     misc_feature    5165..5401
                     /label=NS1 monobody C-terminal part (residues 19-97)
                     /note="NS1 monobody C-terminal part (residues 19-97)"
     misc_feature    5408..5509
                     /label=linker
                     /note="linker"
     misc_feature    5510..5716
                     /label=Aga2p anchor protein
                     /note="Aga2p anchor protein"
     CDS             5738..5971
                     /label=ACP
                     /note="acyl carrier protein (ACP) from E. coli"
     misc_feature    5975..6004
                     /label=Myc-tag
                     /note="Myc-tag"
     regulatory      6018..6324
                     /label=MFa1
                     /note="MFa1"
                     /regulatory_class="terminator"
     promoter        complement(6339..6357)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6364..6380)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      6525..6980
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(7080..7751)
                     /label=TRP1
                     /note="phosphoribosylanthranilate isomerase, required for 
                     tryptophan biosynthesis"
     promoter        complement(7752..8032)
                     /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 ccgaagcatc
     3901 ttcagtacca accaagctgg aagtcgtagc cgcaactttt aaaacaacaa cttctgttat
     3961 tgatactact aatgatgccc aaaatctttt gactcaagcg caaactattg tcaataccct
     4021 taaagattat tgccctatct tgatcgcaaa atctagtagt agcaatggtg gcacaaacaa
     4081 tgctaacacc ccttcatggc aaacagccgg tggcggcaaa aattcatgcg ctacttttgg
     4141 tgcagagttt agtgccgctt cagacatgat taataatgca caaaaaatcg ttcaagaaac
     4201 ccaacaactg agcgccaacc aaccaaaaaa tatcacacaa cctcataatc tgaaccttaa
     4261 cagccctagc agtcttaccg ctttagctca aaaaatgctg aaaaacgcgc aatctcaagc
     4321 agaaatttta aaacttgcca atcaagtgga gagcgatttt aacaaacttt cttcaggcca
     4381 tcttaaagac tacattggga aatgtgacgc ctctgctatc agtagtgcta atatgacaat
     4441 gcagaatcag aagaacaact gggggaacgg gtgcgctggc gtggaagaaa ctcagtcact
     4501 gttaaaaaca agcgccgctg attttaacaa ccaaactcct caaatcaatc aagctcaaaa
     4561 cctggccaac acccttattc aagaacttgg caacaaccct tttcgtgcta gcggtggagg
     4621 atcaggaggt ggaggctcag gtaaactttc agacacttat gaacagctaa gccgtctttt
     4681 aaccaacgat aatggcacaa actcaaagac aagcgcccaa gctatcaacc aagcggttaa
     4741 taatttgaac gaacgtgcaa aaactttagc cggtgggaca accaattctc cggcatatca
     4801 agccacgctt ttagccttac gttctgtgtt agggctgtgg aatagcatgg gttatgctgt
     4861 catctgcgga ggttatacca aaagtccagg cgaaaacaat caaaaagatt tccactacac
     4921 cgatgagaat ggcaacggca ctacaatcaa ttgcggtggg agcacaaata gtaatggcac
     4981 tcatagttat aatggcacaa atacattaaa agcagacaaa aatgtttctc tttctattga
     5041 gcaatatgaa aaaatccatg aagcctatca gattctttca aaagccttaa aacaagccgg
     5101 gcttgctcct ttaaatagca aaggggaaaa gttagaagca catgtaacca catcaaagta
     5161 tgggagcttg cttatcagtt gggatgctcc tgctgtgaca gttgattatt acgtcatcac
     5221 ctacggggaa accggaggga actcacctgt ccagaaattc gaggtaccag gtagtaagag
     5281 taccgcaact atcagtgggt tgaagccagg tgtagactat accataactg tgtatgcttg
     5341 gggttggcac ggtcaagtct attactatat gggttcacca ataagcatta attatcgtac
     5401 aagcttggga ggtggctctg gtggaggggg atcaggaggt ggtggctccg gtggaggtgg
     5461 ttcgggcgga ggtggttccg gtggaggagg ttctggtgga ggcggtagcc aggaactgac
     5521 aactatatgc gagcaaatcc cctcaccaac tttagaatcg acaccatact ctttgtcaac
     5581 gactactatt ttagccaacg ggaaggcaat gcaaggagtt tttgaatatt acaaatcagt
     5641 aacgtttgtc agtaattgcg gttctcaccc ctcaacaact agcaaaggca gccccataaa
     5701 cacacagtat gtttttaagg acaatagctc gacaagtatg agcactatcg aagaacgtgt
     5761 taagaaaatt atcggcgaac agctgggcgt taagcaggaa gaagttacca acaatgcttc
     5821 tttcgttgaa gacctgggcg ccgattctct tgacaccgtt gagctggtaa tggctctgga
     5881 agaagagttt gatactgaga ttccagacga agaagctgag aaaatcacca ccgttcaggc
     5941 tgccattgat tacatcaacg gccaccaggc aagcgaacaa aagttaattt ctgaagagga
     6001 cttgtaatag catgcagatc tgataacaac agtgtagatg taacaaaatc gactttgttc
     6061 ccactgtact tttagctcgt acaaaataca atatactttt catttctccg taaacaacat
     6121 gttttcccat gtaatatcct tttctatttt tcgttccgtt accaacttta cacatacttt
     6181 atatagctat tcacttctat acactaaaaa actaagacaa ttttaatttt gctgcctgcc
     6241 atatttcaat ttgttataaa ttcctataat ttatcctatt agtagctaaa aaaagatgaa
     6301 tgtgaatcga atcctaagag aattgagctc caattcgccc tatagtgagt cgtattacaa
     6361 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     6421 tcgccttgca gcacatcccc ccttcgccag ctggcgtaat agcgaagagg cccgcaccga
     6481 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcgacgcgc cctgtagcgg
     6541 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     6601 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     6661 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     6721 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6781 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6841 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     6901 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     6961 aatattaacg tttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     7021 tcacaccgca ggcaagtgca caaacaatac ttaaataaat actactcagt aataacctat
     7081 ttcttagcat ttttgacgaa atttgctatt ttgttagagt cttttacacc atttgtctcc
     7141 acacctccgc ttacatcaac accaataacg ccatttaatc taagcgcatc accaacattt
     7201 tctggcgtca gtccaccagc taacataaaa tgtaaacttt cggggctctc ttgccttcca
     7261 acccagtcag aaatcgagtt ccaatccaaa agttcacctg tcccacctgc ttctgaatca
     7321 aacaagggaa taaacgaatg aggtttctgt gaagctgcac tgagtagtat gttgcagtct
     7381 tttggaaata cgagtctttt aataactggc aaaccgagga actcttggta ttcttgccac
     7441 gactcatctc catgcagttg gacgatatca atgccgtaat cattgaccag agccaaaaca
     7501 tcctccttag gttgattacg aaacacgcca accaagtatt tcggagtgcc tgaactattt
     7561 ttatatgctt ttacaagact tgaaattttc cttgcaataa ccgggtcaat tgttctcttt
     7621 ctattgggca cacatataat acccagcaag tcagcatcgg aatctagagc acattctgcg
     7681 gcctctgtgc tctgcaagcc gcaaactttc accaatggac cagaactacc tgtgaaatta
     7741 ataacagaca tactccaagc tgcctttgtg tgcttaatca cgtatactca cgtgctcaat
     7801 agtcaccaat gccctccctc ttggccctct ccttttcttt tttcgaccga attaattctt
     7861 aatcggcaaa aaaagaaaag ctccggatca agattgtacg taaggtgaca agctattttt
     7921 caataaagaa tatcttccac tactgccatc tggcgtcata actgcaaagt acacatatat
     7981 tacgatgctg tctattaaat gcttcctata ttatatatat agtaatgtcg tttatggtgc
     8041 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     8101 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     8161 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcga
//
