LOCUS       sequence-148342-        4318 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 4318)
  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 4318)
  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: pAB031a
FEATURES             Location/Qualifiers
     source          1..4318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..915
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(841..860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1001..1023
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1017..1208)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    1426..1447
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1462..1467,1468..1485,1486..1492)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1500..1516
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     CDS             1861..1878
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2260..3051)
                     /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(3052..3123)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3153..4235)
                     /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     4197..4216
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgagattct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accacctctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
      841 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
      901 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
      961 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     1021 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     1081 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     1141 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     1201 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     1261 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacaagagga catccggtca
     1321 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttgctgagg agacttaggg
     1381 accctggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     1441 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     1501 tgtgagcgga taacaatttc acacacctgc aggtgcagta aggaggaaaa aaaaatgggc
     1561 agctctgtga gtagcgttcc gaccaaactg gaagtggttg cagcaacccc gacgagcctg
     1621 ctgatttctt gggatgcccc gatgtctagt agctctgtgt attactatcg tatcacctac
     1681 ggtgaaacgg gcggtaacag cccggtgcag gaatttacgg ttccgtatag tagctctacc
     1741 gcgacgatta gtggcctgag cccgggtgtg gattacacca tcacggttta tgcatggggc
     1801 gaagatagcg cgggttacat gttcatgtat tctccgatta gtatcaatta ccgcacctgc
     1861 caccatcatc accatcatgg cggtagcggc ggtagcaaga caccccccca tgaacgccca
     1921 tatgcttgcc ctgtcgagtc ctgcgatcgc cgcttttctc gctcggatga gcttacccgc
     1981 catatccgca tccacacagg ccagaagccc ttccagtgtc gaatctgcat gcgtaacttc
     2041 agtcgtagtg accaccttac cacccacatc cgcacccaca caggcgagaa gccttttgcc
     2101 tgtgacattt gtgggaggaa gtttgccagg agtgatgaac gcaagaggca taccaaaatc
     2161 catttaagac agaaggacta acttaattaa cggcactcct cagcaaatat aatgaccctc
     2221 ttgataaccc aagagggcat tttttaatgc ccatggcgtt tatttgccga ctaccttggt
     2281 gatctcgcct ttcacgtagt ggacaaattc ttccaactga tctgcgcgcg aggccaagcg
     2341 atcttcttct tgtccaagat aagcctgtct agcttcaagt atgacgggct gatactgggc
     2401 cggcaggcgc tccattgccc agtcggcagc gacatccttc ggcgcgattt tgccggttac
     2461 tgcgctgtac caaatgcggg acaacgtaag cactacattt cgctcatcgc cagcccagtc
     2521 gggcggcgag ttccatagcg ttaaggtttc atttagcgcc tcaaatagat cctgttcagg
     2581 aaccggatca aagagttcct ccgccgctgg acctaccaag gcaacgctat gttctcttgc
     2641 ttttgtcagc aagatagcca gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag
     2701 aatgtcattg cgctgccatt ctccaaattg cagttcgcgc ttagctggat aacgccacgg
     2761 aatgatgtcg tcgtgcacaa caatggtgac ttctacagcg cggagaatct cgctctctcc
     2821 aggggaagcc gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag
     2881 ccttacggtc accgtaacca gcaaatcaat atcactgtgt ggcttcaggc cgccatccac
     2941 tgcggagccg tacaaatgta cggccagcaa cgtcggttcg agatggcgct cgatgacgcc
     3001 aactacctct gatagttgag tcgatacttc ggcgatcacc gcttccctca tactcttcct
     3061 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3121 atgtatttag aaaaatatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3181 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3241 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     3301 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     3361 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     3421 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     3481 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     3541 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     3601 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     3661 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     3721 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     3781 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     3841 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     3901 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     3961 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     4021 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     4081 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     4141 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     4201 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     4261 tccgggcgct atcatgccat accgcgaaag gttttgcacc ataggccaaa taggccgt
//
