LOCUS       sequence-148343-        5556 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 5556)
  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 5556)
  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: pAB043a
FEATURES             Location/Qualifiers
     source          1..5556
                     /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             3399..3416
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3498..4289)
                     /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(4290..4361)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(4391..5473)
                     /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     5435..5454
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      join(5556,1..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 atgacaagaa gatcgaagaa ggcaagttag tgatttggat caacggcgat aaaggctata
     2341 acggtttagc ggaagtaggc aagaagtttg agaaggatac cggaatcaaa gtgacggtcg
     2401 aacacccgga taagttagag gagaagttcc ctcaagtagc ggcaacgggc gacggtcctg
     2461 acatcatctt ttgggcacac gaccgcttcg gtggttacgc tcagtcaggt ttattagcgg
     2521 agattactcc agacaaagcg ttccaggaca agttatatcc atttacgtgg gacgcagtgc
     2581 gttataacgg caagttaatc gcatatccca ttgcggtaga agccctgagc ctgatttaca
     2641 acaaggacct gttacccaac cctccgaaga cgtgggagga gattccggct ttagataagg
     2701 aactgaaagc gaaaggcaag tctgccctga tgtttaacct gcaagaaccc tacttcacct
     2761 ggcctctgat cgcggcggat ggcggttatg cgtttaagta cgaaaacggc aaatacgata
     2821 ttaaggatgt aggcgtcgat aacgccggtg ccaaagccgg tctgaccttt ttagttgatc
     2881 tgattaagaa taagcacatg aacgcagaca ccgactacag cattgcggaa gctgcgttta
     2941 ataaaggtga gaccgccatg acgattaacg gaccttgggc gtggtcgaac attgatacca
     3001 gtaaagtcaa ttacggcgtt acagtcctgc cgaccttcaa aggtcagccg tcaaaaccgt
     3061 tcgtaggtgt cttatccgcc ggtatcaacg cggcgtcccc aaataaagag ttggctaaag
     3121 agttcctgga aaactatctg ctgacagacg aaggactgga ggctgtgaac aaggacaagc
     3181 cactgggcgc tgttgcgctg aaaagttatg aggaagaact ggcgaaagat ccccgcatcg
     3241 cggcgacgat ggaaaacgcc caaaaaggcg aaatcatgcc gaacattccg cagatgtcag
     3301 ctttttggta tgccgtacgc acggctgtta ttaacgccgc gtcgggccgc caaaccgttg
     3361 atgaggcact gaaggacgcg cagactcgta tcaccaagca ccatcatcac catcattaac
     3421 ttaattaacg gcactcctca gcaaatataa tgaccctctt gataacccaa gagggcattt
     3481 tttaatgccc atggcgttta tttgccgact accttggtga tctcgccttt cacgtagtgg
     3541 acaaattctt ccaactgatc tgcgcgcgag gccaagcgat cttcttcttg tccaagataa
     3601 gcctgtctag cttcaagtat gacgggctga tactgggccg gcaggcgctc cattgcccag
     3661 tcggcagcga catccttcgg cgcgattttg ccggttactg cgctgtacca aatgcgggac
     3721 aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt
     3781 aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc
     3841 gccgctggac ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa gatagccaga
     3901 tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg ctgccattct
     3961 ccaaattgca gttcgcgctt agctggataa cgccacggaa tgatgtcgtc gtgcacaaca
     4021 atggtgactt ctacagcgcg gagaatctcg ctctctccag gggaagccga agtttccaaa
     4081 aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc
     4141 aaatcaatat cactgtgtgg cttcaggccg ccatccactg cggagccgta caaatgtacg
     4201 gccagcaacg tcggttcgag atggcgctcg atgacgccaa ctacctctga tagttgagtc
     4261 gatacttcgg cgatcaccgc ttccctcata ctcttccttt ttcaatatta ttgaagcatt
     4321 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaatattaat
     4381 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4441 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4501 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4561 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4621 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     4681 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4741 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     4801 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4861 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4921 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4981 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     5041 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     5101 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     5161 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     5221 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     5281 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     5341 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     5401 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5461 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     5521 cgcgaaaggt tttgcaccat aggccaaata ggccgt
//
