LOCUS       62056_18110        8011 bp DNA     circular SYN 07-JAN-2021
DEFINITION  Yeast display vector pNMB2, complete sequence.
ACCESSION   MT338523
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8011)
  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 8011)
  AUTHORS   Steyaert J, Uchanski T, Pardon E.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-APR-2020) VIB-VUB Center for Structural Biology, VIB, 
            Pleinlaan 2, Brussels 1050, Belgium
REFERENCE   3  (bases 1 to 8011)
  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 
            (14-APR-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..8011
                     /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=nanobody beta-strain A (residues 1-14)
                     /note="nanobody beta-strain A (residues 1-14)"
     misc_feature    3938..5161
                     /label=cHopQ scaffold protein
                     /note="cHopQ scaffold protein"
     misc_feature    5162..5200
                     /label=multi cloning site
                     /note="multi cloning site"
     misc_feature    5201..5302
                     /label=linker
                     /note="linker"
     misc_feature    5303..5509
                     /label=Aga2p anchor protein
                     /note="Aga2p anchor protein"
     CDS             5531..5764
                     /label=ACP
                     /note="acyl carrier protein (ACP) from E. coli"
     misc_feature    5768..5797
                     /label=Myc-tag
                     /note="Myc-tag"
     regulatory      5811..6117
                     /label=MFa1
                     /note="MFa1"
                     /regulatory_class="terminator"
     promoter        complement(6132..6150)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6157..6173)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      6318..6773
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(6873..7544)
                     /label=TRP1
                     /note="phosphoribosylanthranilate isomerase, required for 
                     tryptophan biosynthesis"
     promoter        complement(7545..7825)
                     /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 gagcaagata
      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 ggaagagcgc
     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 agtacagact 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 tggatccgga agagcttagt cgacaagctc ttcaagcttg ggaggtggct ctggtggagg
     5221 gggatcagga ggtggtggct ccggtggagg tggttcgggc ggaggtggtt ccggtggagg
     5281 aggttctggt ggaggcggta gccaggaact gacaactata tgcgagcaaa tcccctcacc
     5341 aactttagaa tcgacaccat actctttgtc aacgactact attttagcca acgggaaggc
     5401 aatgcaagga gtttttgaat attacaaatc agtaacgttt gtcagtaatt gcggttctca
     5461 cccctcaaca actagcaaag gcagccccat aaacacacag tatgttttta aggacaatag
     5521 ctcgacaagt atgagcacta tcgaagaacg tgttaagaaa attatcggcg aacagctggg
     5581 cgttaagcag gaagaagtta ccaacaatgc ttctttcgtt gaagacctgg gcgccgattc
     5641 tcttgacacc gttgagctgg taatggctct ggaagaagag tttgatactg agattccaga
     5701 cgaagaagct gagaaaatca ccaccgttca ggctgccatt gattacatca acggccacca
     5761 ggcaagcgaa caaaagttaa tttctgaaga ggacttgtaa tagcatgcag atctgataac
     5821 aacagtgtag atgtaacaaa atcgactttg ttcccactgt acttttagct cgtacaaaat
     5881 acaatatact tttcatttct ccgtaaacaa catgttttcc catgtaatat ccttttctat
     5941 ttttcgttcc gttaccaact ttacacatac tttatatagc tattcacttc tatacactaa
     6001 aaaactaaga caattttaat tttgctgcct gccatatttc aatttgttat aaattcctat
     6061 aatttatcct attagtagct aaaaaaagat gaatgtgaat cgaatcctaa gagaattgag
     6121 ctccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg
     6181 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccccttcgc
     6241 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     6301 gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     6361 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     6421 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     6481 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     6541 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     6601 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     6661 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     6721 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa tttcctgatg
     6781 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaggcaagt gcacaaacaa
     6841 tacttaaata aatactactc agtaataacc tatttcttag catttttgac gaaatttgct
     6901 attttgttag agtcttttac accatttgtc tccacacctc cgcttacatc aacaccaata
     6961 acgccattta atctaagcgc atcaccaaca ttttctggcg tcagtccacc agctaacata
     7021 aaatgtaaac tttcggggct ctcttgcctt ccaacccagt cagaaatcga gttccaatcc
     7081 aaaagttcac ctgtcccacc tgcttctgaa tcaaacaagg gaataaacga atgaggtttc
     7141 tgtgaagctg cactgagtag tatgttgcag tcttttggaa atacgagtct tttaataact
     7201 ggcaaaccga ggaactcttg gtattcttgc cacgactcat ctccatgcag ttggacgata
     7261 tcaatgccgt aatcattgac cagagccaaa acatcctcct taggttgatt acgaaacacg
     7321 ccaaccaagt atttcggagt gcctgaacta tttttatatg cttttacaag acttgaaatt
     7381 ttccttgcaa taaccgggtc aattgttctc tttctattgg gcacacatat aatacccagc
     7441 aagtcagcat cggaatctag agcacattct gcggcctctg tgctctgcaa gccgcaaact
     7501 ttcaccaatg gaccagaact acctgtgaaa ttaataacag acatactcca agctgccttt
     7561 gtgtgcttaa tcacgtatac tcacgtgctc aatagtcacc aatgccctcc ctcttggccc
     7621 tctccttttc ttttttcgac cgaattaatt cttaatcggc aaaaaaagaa aagctccgga
     7681 tcaagattgt acgtaaggtg acaagctatt tttcaataaa gaatatcttc cactactgcc
     7741 atctggcgtc ataactgcaa agtacacata tattacgatg ctgtctatta aatgcttcct
     7801 atattatata tatagtaatg tcgtttatgg tgcactctca gtacaatctg ctctgatgcc
     7861 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     7921 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     7981 aggttttcac cgtcatcacc gaaacgcgcg a
//
