LOCUS       sequence-148351-        3998 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 3998)
  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 3998)
  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: pAB061i3
FEATURES             Location/Qualifiers
     source          1..3998
                     /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(1940..2731)
                     /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(2732..2803)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(2833..3915)
                     /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     3877..3896
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      join(3998,1..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 aggacttgta
     1861 acttaattaa cggcactcct cagcaaatat aatgaccctc ttgataaccc aagagggcat
     1921 tttttaatgc ccatggcgtt tatttgccga ctaccttggt gatctcgcct ttcacgtagt
     1981 ggacaaattc ttccaactga tctgcgcgcg aggccaagcg atcttcttct tgtccaagat
     2041 aagcctgtct agcttcaagt atgacgggct gatactgggc cggcaggcgc tccattgccc
     2101 agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac caaatgcggg
     2161 acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag ttccatagcg
     2221 ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca aagagttcct
     2281 ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc aagatagcca
     2341 gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg cgctgccatt
     2401 ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg tcgtgcacaa
     2461 caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc gaagtttcca
     2521 aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacggtc accgtaacca
     2581 gcaaatcaat atcactgtgt ggcttcaggc cgccatccac tgcggagccg tacaaatgta
     2641 cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct gatagttgag
     2701 tcgatacttc ggcgatcacc gcttccctca tactcttcct ttttcaatat tattgaagca
     2761 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaatatta
     2821 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     2881 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     2941 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     3001 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     3061 tggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     3121 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     3181 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     3241 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     3301 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     3361 taacagtgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     3421 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     3481 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     3541 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     3601 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     3661 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     3721 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     3781 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     3841 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     3901 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     3961 accgcgaaag gttttgcacc ataggccaaa taggccgt
//
