LOCUS       62056_16745        5754 bp DNA     circular SYN 07-JAN-2021
DEFINITION  Cloning vector pMESD2, complete sequence.
ACCESSION   MT328400
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5754)
  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 5754)
  AUTHORS   Steyaert J, Uchanski T, Pardon E.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-APR-2020) VIB-VUB Center for Structural Biology, VIB, 
            Pleinlaan 2, Brussels 1050, Belgium
REFERENCE   3  (bases 1 to 5754)
  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 
            (10-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..5754
                     /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)."
     misc_feature    225..281
                     /label=DsbA signal peptide
                     /note="DsbA signal peptide"
     misc_feature    282..320
                     /label=nanobody beta-strain A (residues 1-14)
                     /note="nanobody beta-strain A (residues 1-14)"
     misc_feature    321..1544
                     /label=cHopQ scaffold protein
                     /note="cHopQ scaffold protein"
     misc_feature    1545..1547
                     /label=nanobody residue 17
                     /note="nanobody residue 17"
     misc_feature    1548..1580
                     /label=multi cloning site
                     /note="multi cloning site"
     CDS             1581..1598
                     /label=6xHis
                     /note="6xHis affinity tag"
     misc_feature    1599..1610
                     /label=EPEA-tag
                     /note="EPEA-tag"
     misc_feature    1614..1643
                     /label=HA-tag
                     /note="HA-tag"
     gene            1644..2877
                     /gene="M13 geneIII"
                     /label=M13 geneIII
     primer_bind     complement(2883..2899)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      3112..3567
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3849..3953
                     /label=AmpR promoter
     CDS             3954..4811
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      4985..5573
                     /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 aagatttggc
      241 tggcgctggc tggtttagtt ttagcgttta gcgcatcggc gcaggtgcag ctggtggagt
      301 ctgggggagg actagtacag actaaaacaa caacttctgt tattgatact actaatgatg
      361 cccaaaatct tttgactcaa gcgcaaacta ttgtcaatac ccttaaagat tattgcccta
      421 tcttgatcgc aaaatctagt agtagcaatg gtggcacaaa caatgctaac accccttcat
      481 ggcaaacagc cggtggcggc aaaaattcat gcgctacttt tggtgcagag tttagtgccg
      541 cttcagacat gattaataat gcacaaaaaa tcgttcaaga aacccaacaa ctgagcgcca
      601 accaaccaaa aaatatcaca caacctcata atctgaacct taacagccct agcagtctta
      661 ccgctttagc tcaaaaaatg ctgaaaaacg cgcaatctca agcagaaatt ttaaaacttg
      721 ccaatcaagt ggagagcgat tttaacaaac tttcttcagg ccatcttaaa gactacattg
      781 ggaaatgtga cgcctctgct atcagtagtg ctaatatgac aatgcagaat cagaagaaca
      841 actgggggaa cgggtgcgct ggcgtggaag aaactcagtc actgttaaaa acaagcgccg
      901 ctgattttaa caaccaaact cctcaaatca atcaagctca aaacctggcc aacaccctta
      961 ttcaagaact tggcaacaac ccttttcgtg ctagcggtgg aggatcagga ggtggaggct
     1021 caggtaaact ttcagacact tatgaacagc taagccgtct tttaaccaac gataatggca
     1081 caaactcaaa gacaagcgcc caagctatca accaagcggt taataatttg aacgaacgtg
     1141 caaaaacttt agccggtggg acaaccaatt ctccggcata tcaagccacg cttttagcct
     1201 tacgttctgt gttagggctg tggaatagca tgggttatgc tgtcatctgc ggaggttata
     1261 ccaaaagtcc aggcgaaaac aatcaaaaag atttccacta caccgatgag aatggcaacg
     1321 gcactacaat caattgcggt gggagcacaa atagtaatgg cactcatagt tataatggca
     1381 caaatacatt aaaagcagac aaaaatgttt ctctttctat tgagcaatat gaaaaaatcc
     1441 atgaagccta tcagattctt tcaaaagcct taaaacaagc cgggcttgct cctttaaata
     1501 gcaaagggga aaagttagaa gcacatgtaa ccacatcaaa gtatggatcc ggaagagctt
     1561 agtcgacaag ctcttcaagt caccaccatc accatcacga acctgaagcc tagtacccgt
     1621 acgacgttcc ggactacggt tccactgttg aaagttgttt agcaaaacct catacagaaa
     1681 attcatttac taacgtctgg aaagacgaca aaactttaga tcgttacgct aactatgagg
     1741 gctgtctgtg gaatgctaca ggcgttgtcg tttgtactgg tgacgaaact cagtgttacg
     1801 gtacatgggt tcctattggg cttgctatcc ctgaaaatga gggtggtggc tctgagggtg
     1861 gcggttctga gggtggcggt tctgagggtg gcggtactaa acctcctgag tacggtgata
     1921 cacctattcc gggctatact tatatcaacc ctctcgacgg cacttatccg cctggtactg
     1981 agcaaaaccc cgctaatcct aatccttctc ttgaggagtc tcagcctctt aatactttca
     2041 tgtttcagaa taataggttc cgaaataggc agggtgcatt aactgtttat acgggcactg
     2101 ttactcaagg cactgacccc gttaaaactt attaccagta cactcctgta tcatcaaaag
     2161 ccatgtatga cgcttactgg aacggtaaat tcagagactg cgctttccat tctggcttta
     2221 atgaggatcc attcgtttgt gaatatcaag gccaatcgtc tgacctgcct caacctcctg
     2281 tcaatgctgg cggcggctct ggtggtggtt ctggtggcgg ctctgagggt ggcggctctg
     2341 agggtggcgg ctctgagggt ggcggttctg agggtggcgg ctctgagggt ggcggttccg
     2401 gtggcggctc cggttccggt gattttgatt atgaaaaaat ggcaaacgct aataaggggg
     2461 ctatgaccga aaatgccgat gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt
     2521 ctgtcgctac tgattacggt gctgctatcg atggtttcat tggtgacgtt tccggccttg
     2581 ctaatggtaa tggtgctact ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg
     2641 gtgacggtga taattcacct ttaatgaata atttccgtca atatttacct tctttacctc
     2701 agtcggttga atgtcgccct tatgtctttg gcgctggtaa accatatgaa ttttctattg
     2761 attgtgacaa aataaactta ttccgtggtg tctttgcgtt tcttttatat gttgccacct
     2821 ttatgtatgt attttcgacg tttgctaaca tactgcgtaa taaggagtct taataagaat
     2881 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     2941 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     3001 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     3061 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     3121 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3181 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3241 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3301 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     3361 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3421 tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta taagggattt
     3481 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3541 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     3601 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3661 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     3721 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     3781 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     3841 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3901 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     3961 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4021 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4081 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4141 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4201 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4261 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4321 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4381 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4441 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     4501 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4561 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4621 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4681 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4741 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4801 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4861 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4921 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4981 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5041 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5101 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     5161 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5221 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5281 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5341 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5401 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5461 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5521 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5581 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5641 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     5701 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaag
//
