LOCUS       sequence-148350-        4301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Directed Evolution of Protein-Protein Interactions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4301)
  AUTHORS   Badran AH, Guzov VM, Huai Q, Kemp MM, Vishwanath P, Kain W, Nance 
            AM, Evdokimov A, Moshiri F, Turner KH, Wang P, Malvar T, Liu DR
  TITLE     Continuous evolution of Bacillus thuringiensis toxins overcomes 
            insect resistance.
  JOURNAL   Nature. 2016 May 5;533(7601):58-63. doi: 10.1038/nature17938. Epub 
            2016 Apr 27.
  PUBMED    27120167
REFERENCE   2  (bases 1 to 4301)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nature17938"; journalName: "Nature"; date: "2016-05-5- 5"; 
            volume: "533"; issue: "7601"; pages: "58-63"
COMMENT     Sequence Label: pAB061i2
FEATURES             Location/Qualifiers
     source          1..4301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     489..508
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     742..759
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    774..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             1547..1819
                     /codon_start=1
                     /gene="E. coli rpoZ"
                     /product="DNA-directed RNA polymerase subunit omega"
                     /label=RNAP omega
                     /note="promotes RNA polymerase assembly"
                     /translation="MARVTVQDAVEKIGNRFDLVLVAARRARQMQVGGKDPLVPEENDK
                     TTVIALREIEEGLINNQILDVRERQEQQEQEAAELQAVTAIAEGRR"
     CDS             1829..1858
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(2243..3034)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(3035..3106)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3136..4218)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4180..4199
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      join(4301,1..588)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
       61 gcggtggttt gtttgccgga tcaagagcta ccacctcttt ttccgaaggt aactggcttc
      121 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
      181 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
      241 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
      301 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
      361 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      421 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
      481 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
      541 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
      601 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
      661 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
      721 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
      781 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acaagaggac atccggtcaa
     1321 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttgctgagga ttctagagca
     1381 cagctaacac cacgtcgtcc ctatctgctg ccctaggtct atgagtggtt gctggataac
     1441 tttacgggca tgcataaggc tcggtatcta tattcaggga gaccacaacg gtttccctct
     1501 acaaataatt ttgtttaact tttactagag taaggaggaa aaaaaaatgg cacgcgtaac
     1561 tgttcaggac gctgtagaga aaattggtaa ccgttttgac ctggtactgg tcgccgcgcg
     1621 tcgcgctcgt cagatgcagg taggcggaaa ggatccgctg gtaccggaag aaaacgataa
     1681 aaccactgta atcgcgctgc gcgaaatcga agaaggtctg atcaacaacc agatcctcga
     1741 cgttcgcgaa cgccaggaac agcaagagca ggaagccgct gaattacaag ccgttaccgc
     1801 tattgctgaa ggtcgtcgtg cggccgcgga acaaaagctt atttctgaag aggacttggg
     1861 cagctctgtg agtagcgttc cgaccaaact ggaagtggtt gcagcaaccc cgacgagcct
     1921 gctgatttct tgggatgccc cgatgtctag tagctctgtg tattactatc gtatcaccta
     1981 cggtgaaacg ggcggtaaca gcccggtgca ggaatttacg gttccgtata gtagctctac
     2041 cgcgacgatt agtggcctga gcccgggtgt ggattacacc atcacggttt atgcatgggg
     2101 cgaagatagc gcgggtgcga tgttcatgta ttctccgatt agtatcaatt accgcacctg
     2161 ctaacttaat taacggcact cctcagcaaa tataatgacc ctcttgataa cccaagaggg
     2221 cattttttaa tgcccatggc gtttatttgc cgactacctt ggtgatctcg cctttcacgt
     2281 agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct tcttgtccaa
     2341 gataagcctg tctagcttca agtatgacgg gctgatactg ggccggcagg cgctccattg
     2401 cccagtcggc agcgacatcc ttcggcgcga ttttgccggt tactgcgctg taccaaatgc
     2461 gggacaacgt aagcactaca tttcgctcat cgccagccca gtcgggcggc gagttccata
     2521 gcgttaaggt ttcatttagc gcctcaaata gatcctgttc aggaaccgga tcaaagagtt
     2581 cctccgccgc tggacctacc aaggcaacgc tatgttctct tgcttttgtc agcaagatag
     2641 ccagatcaat gtcgatcgtg gctggctcga agatacctgc aagaatgtca ttgcgctgcc
     2701 attctccaaa ttgcagttcg cgcttagctg gataacgcca cggaatgatg tcgtcgtgca
     2761 caacaatggt gacttctaca gcgcggagaa tctcgctctc tccaggggaa gccgaagttt
     2821 ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg gtcaccgtaa
     2881 ccagcaaatc aatatcactg tgtggcttca ggccgccatc cactgcggag ccgtacaaat
     2941 gtacggccag caacgtcggt tcgagatggc gctcgatgac gccaactacc tctgatagtt
     3001 gagtcgatac ttcggcgatc accgcttccc tcatactctt cctttttcaa tattattgaa
     3061 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     3121 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3181 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     3241 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     3301 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     3361 taatggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     3421 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     3481 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     3541 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     3601 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     3661 cgctaacagt gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     3721 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     3781 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     3841 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     3901 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     3961 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     4021 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     4081 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     4141 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     4201 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     4261 cataccgcga aaggttttgc accataggcc aaataggccg t
//
