LOCUS       sequence-148341-        4746 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 4746)
  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 4746)
  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: pAB029f
FEATURES             Location/Qualifiers
     source          1..4746
                     /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"
     protein_bind    1425..1446
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1461..1466,1467..1484,1485..1491)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1499..1515
                     /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             1554..2264
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     CDS             2265..2288
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2589..2606
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2688..3479)
                     /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(3480..3551)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(3581..4663)
                     /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     4625..4644
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      join(4746,1..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 gacttaggga
     1381 ccctggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     1441 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     1501 gtgagcggat aacaatttca cacacctgca ggtgcagtaa ggaggaaaaa aaaatgagca
     1561 caaaaaagaa accattaaca caagagcagc ttgaggacgc acgtcgcctt aaagcaattt
     1621 atgaaaaaaa gaaaaatgaa cttggcttat cccaggaatc tgtcgcagac aagatgggga
     1681 tggggcagtc aggcgttggt gctttattta atggcatcaa tgcattaaat gcttataacg
     1741 ccgcattgct tgcaaaaatt ctcaaagtta gcgttgaaga atttagccct tcaatcgcca
     1801 gagaaatcta cgagatgtat gaagcggtta gtatgcagcc gtcacttaga agtgagtatg
     1861 agtaccctgt tttttctcat gttcaggcag ggatgttctc acctgagctt agaaccttta
     1921 ccaaaggtga tgcggagaga tgggtaagca caaccaaaaa agccagtgat tctgcattct
     1981 ggcttgaggt tgaaggtaat tccatgaccg caccaacagg ctccaagcca agctttcctg
     2041 acggaatgtt aattctcgtt gaccctgagc aggctgttga gccaggtgat ttctgcatag
     2101 ccagacttgg gggtgatgag tttaccttca agaaactgat cagggatagc ggtcaggtgt
     2161 ttttacaacc actaaaccca cagtacccaa tgatcccatg caatgagagt tgttccgttg
     2221 tggggaaagt tatcgctagt cagtggcctg aagagacgtt tggggactac aaggacgacg
     2281 atgacaagag tctggaaaaa cacagctggt atcatggccc tgtgagccgt aacgcggccg
     2341 aatacctgct gagctctggc attaatggtt cttttctggt tcgtgaaagt gaaagtagcc
     2401 cgggccagcg cagcatttct ctgcgttatg aaggtcgcgt gtatcactac cgtatcaaca
     2461 ccgccagcga tggcaaactg tacgtttcta gtgaatctcg cttcaatacc ctggcagaac
     2521 tggtgcatca ccatagcacg gttgcggatg gtctgatcac cacgctgcat tatccggcgc
     2581 cgaaacgcca ccatcatcac catcattaac ttaattaacg gcactcctca gcaaatataa
     2641 tgaccctctt gataacccaa gagggcattt tttaatgccc atggcgttta tttgccgact
     2701 accttggtga tctcgccttt cacgtagtgg acaaattctt ccaactgatc tgcgcgcgag
     2761 gccaagcgat cttcttcttg tccaagataa gcctgtctag cttcaagtat gacgggctga
     2821 tactgggccg gcaggcgctc cattgcccag tcggcagcga catccttcgg cgcgattttg
     2881 ccggttactg cgctgtacca aatgcgggac aacgtaagca ctacatttcg ctcatcgcca
     2941 gcccagtcgg gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc aaatagatcc
     3001 tgttcaggaa ccggatcaaa gagttcctcc gccgctggac ctaccaaggc aacgctatgt
     3061 tctcttgctt ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg ctcgaagata
     3121 cctgcaagaa tgtcattgcg ctgccattct ccaaattgca gttcgcgctt agctggataa
     3181 cgccacggaa tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg gagaatctcg
     3241 ctctctccag gggaagccga agtttccaaa aggtcgttga tcaaagctcg ccgcgttgtt
     3301 tcatcaagcc ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg cttcaggccg
     3361 ccatccactg cggagccgta caaatgtacg gccagcaacg tcggttcgag atggcgctcg
     3421 atgacgccaa ctacctctga tagttgagtc gatacttcgg cgatcaccgc ttccctcata
     3481 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3541 atatttgaat gtatttagaa aaatattaat tgcgttgcgc tcactgcccg ctttccagtc
     3601 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3661 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     3721 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     3781 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     3841 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     3901 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     3961 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     4021 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     4081 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     4141 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     4201 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     4261 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     4321 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     4381 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     4441 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     4501 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     4561 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     4621 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     4681 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcaccat aggccaaata
     4741 ggccgt
//
