LOCUS       62056_16760        5595 bp DNA     circular SYN 07-JAN-2021
DEFINITION  Cloning vector pMESP23NO, complete sequence.
ACCESSION   MT338522
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5595)
  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 5595)
  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 5595)
  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..5595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     sig_peptide     225..290
                     /label=pelB signal sequence
                     /note="leader peptide for secretion"
     misc_feature    291..326
                     /label=nanobody beta-strain A (residues 1-13)
                     /note="nanobody beta-strain A (residues 1-13)"
     misc_feature    327..2651
                     /label=cYgjK scaffold protein
                     /note="cYgjK scaffold protein"
     misc_feature    2652..2684
                     /label=multi cloning site
                     /note="multi cloning site"
     CDS             2685..2702
                     /label=6xHis
                     /note="6xHis affinity tag"
     misc_feature    2703..2714
                     /label=EPEA-tag
                     /note="EPEA-tag"
     primer_bind     complement(2724..2740)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      2953..3408
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3690..3794
                     /label=AmpR promoter
     CDS             3795..4652
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      4826..5414
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agagcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacaagctta aggagacagt acatatgaaa tacctattgc
      241 ctacggcagc cgctggattg ttattactcg cggcccagcc ggccatggcc caggtgcagc
      301 tggtggagtc tgggggagga ctagtaaaag aagagacgca gtctgggctg aacaactacg
      361 cccgtgtggt ggagaaaggc cagtacgaca gtctggaaat tcctgcacag gttgctgcat
      421 cgtgggaatc ggggcgtgat gatgccgccg tctttgggtt tatcgacaaa gaacagttgg
      481 ataaatatgt cgctaatggc ggcaaacgta gcgactggac agtgaaattc gccgaaaacc
      541 gtagtcagga cggaacattg ctgggctact cgcttttgca ggagtcggtg gatcaggcca
      601 gctatatgta cagcgataac cattatctgg cagagatggc aaccatcctg ggtaagccag
      661 aagaggccaa acgttatcgt cagttggcac agcagctggc agactacatc aacacctgta
      721 tgttcgaccc tactacccag ttctactatg acgtacgtat tgaagataaa ccactggcta
      781 acggctgcgc aggcaaacct attgttgagc gtggtaaagg gccagaaggc tggtcgccac
      841 tgtttaacgg tgcagcaact caggccaatg ccgacgctgt ggtgaaggtg atgcttgatc
      901 ctaaagagtt caacaccttt gtcccactgg gaactgccgc tttaaccaat cctgcttttg
      961 gcgctgacat ctactggcgt gggcgtgtat gggtggatca gttctggttt ggtctgaaag
     1021 ggatggagcg ttacggttat cgtgatgatg ccctgaagct ggcagataca ttcttccggc
     1081 acgccaaagg gttaaccgcc gatggaccaa ttcaggaaaa ttacaaccca ctgacaggcg
     1141 cacagcaagg cgcaccaaat ttctcctgga gcgctgctca tttgtatatg ttgtataacg
     1201 attttttccg taagcaagct agcggtggag gatcaggagg tggaggatca ggaggtggag
     1261 gctcaggtaa cgccgacaat tataaaaacg tgattaaccg tactggggcc cctcagtaca
     1321 tgaaggatta cgattacgac gatcaccagc gttttaatcc atttttcgat cttggagcct
     1381 ggcatggtca tctgttgcca gacggcccta acactatggg cggctttcca ggcgttgctc
     1441 tgctgacaga agagtacatc aactttatgg caagcaattt cgaccgtctg accgtctggc
     1501 aggacggcaa gaaagtagac tttacactgg aggcatacag tattcctggt gctctggtgc
     1561 aaaaactgac agcaaaagat gtgcaagtcg aaatgactct gcgtttcgcc actcctcgta
     1621 cctcacttct ggaaaccaaa atcaccagca ataaaccact ggatctggtg tgggacggcg
     1681 aactgctgga aaaactggaa gctaaagaag ggaaacctct ttccgataaa accattgctg
     1741 gcgaataccc tgactatcag cgtaaaatca gcgccacccg tgatggcctg aaagtcacct
     1801 ttggcaaagt gcgtgccacc tgggatctgc tgacctccgg cgaatcggaa tatcaggtgc
     1861 ataaatccct gcctgtgcag actgaaatca acggtaatcg ttttaccagt aaggcccata
     1921 tcaacggttc gaccacactt tacaccacct attcccatct gctgaccgca caggaagtta
     1981 gcaaagagca aatgcagatc cgtgatattc tggctcgtcc agcattttat ctgaccgcct
     2041 cgcagcaacg ttgggaagaa tacctgaaga aagggttaac caatccagat gcaacccctg
     2101 aacagactcg tgtcgctgtg aaagcaatcg aaactctgaa cggtaactgg cgttcacctg
     2161 gcggtgctgt gaaatttaac accgttacac catcggtgac agggcgttgg ttctccggca
     2221 atcagacctg gccttgggat acctggaagc aggcttttgc tatggcacat ttcaatccag
     2281 acatcgccaa agagaatatc cgtgctgtct tctcctggca gatccagcct ggcgatagcg
     2341 tgcgtccaca ggatgtgggc tttgtcccag acctgattgc ttggaacctg agtccagagc
     2401 gtggtggcga tggcggcaac tggaacgaac gtaataccaa acctagcctt gccgcctggt
     2461 cggtgatgga agtgtataac gtcacccaag ataaaacctg ggtggcagag atgtacccta
     2521 aactggtggc ctatcacgac tggtggttac gtaaccgtga tcacaacggc aacggcgtgc
     2581 cagaatatgg cgcaacccgt gacaaagccc acaacactga gagcggcgag atgctgttta
     2641 cagtgaaaaa gtccggaaga gcttagtcga caagctcttc aagtcaccac catcaccatc
     2701 acgaacctga agcctaagaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     2761 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     2821 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     2881 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     2941 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3001 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3061 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3121 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     3181 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3241 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt
     3301 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3361 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     3421 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     3481 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3541 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     3601 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     3661 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     3721 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     3781 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     3841 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     3901 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     3961 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4021 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4081 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4141 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4201 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4261 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     4321 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     4381 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     4441 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     4501 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     4561 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     4621 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     4681 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     4741 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     4801 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4861 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4921 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     4981 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5041 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5101 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5161 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5221 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5281 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5341 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5401 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5461 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     5521 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     5581 gcgaggaagc ggaag
//
