LOCUS       sequence-148330-        7161 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 7161)
  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 7161)
  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: pAB085d
FEATURES             Location/Qualifiers
     source          1..7161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..951)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      999..1221
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             1888..2967
                     /codon_start=1
                     /gene="luxA"
                     /product="LuxA luciferase subunit"
                     /label=LuxA
                     /note="from the Photorhabdus luminescens bioluminescence 
                     operon"
                     /translation="MKFGNFLLTYQPPQFSQTEVMKRLVKLGRISEECGFDTVWLLEHH
                     FTEFGLLGNPYVAAAYLLGATKKLNVGTAAIVLPTAHPVRQLEDVNLLDQMSKGRFRFG
                     ICRGLYNKDFRVFGTDMNNSRALAECWYGLIKNGMTEGYMEADNEHIKFHKVKVNPAAY
                     SRGGAPVYVVAESASTTEWAAQFGLPMILSWIINTNEKKAQLELYNEVAQEYGHDIHNI
                     DHCLSYITSVDHDSIKAKEICRKFLGHWYDSYVNATTIFDDSDQTRGYDFNKGQWRDFV
                     LKGHKDTNRRIDYSYEINPVGTPQECIDIIQKDIDATGISNICCGFEANGTVDEIIASM
                     KLFQSDVMPFLKEKQRSLLY"
     CDS             3031..4011
                     /codon_start=1
                     /gene="luxB"
                     /product="LuxB luciferase subunit"
                     /label=LuxB
                     /note="from the Photorhabdus luminescens bioluminescence 
                     operon"
                     /translation="KFGLFFLNFINSTTVQEQSIVRMQEITEYVDKLNFEQILVYENHF
                     SDNGVVGAPLTVSGFLLGLTEKIKIGSLNHIITTHHPVRIAEEACLLDQLSEGRFILGF
                     SDCEKKDEMHFFNRPVEYQQQLFEECYEIINDALTTGYCNPDNDFYSFPKISVNPHAYT
                     PGGPRKYVTATSHHIVEWAAKKGIPLIFKWDDSNDVRYEYAERYKAVADKYDVDLSEID
                     HQLMILVNYNEDSNKAKQETRAFISDYVLEMHPNENFENKLEEIIAENAVGNYTECITA
                     AKLAIEKCGAKSVLLSFEPMNDLMSQKNVINIVDDNIKKYHTEYT"
     CDS             complement(4782..4805)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        5506..5560
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    5507..5525
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5537..5555
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    5561..5579
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      complement(5669..5696)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(5788..5874)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     6027..6044
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(join(6090..6881,6882..6950))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6713..6732
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6951..7055)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7123..7141
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg gttcgttgtt tttgcgtgag
       61 ccatgagaac gaaccattga gatcatgctt actttgcatg tcactcaaaa attttgcctc
      121 aaaactggtg agctgaattt ttgcagttaa agcatcgtgt agtgtttttc ttagtccgtt
      181 acgtaggtag gaatctgatg taatggttgt tggtattttg tcaccattca tttttatctg
      241 gttgttctca agttcggtta cgagatccat ttgtctatct agttcaactt ggaaaatcaa
      301 cgtatcagtc gggcggcctc gcttatcaac caccaatttc atattgctgt aagtgtttaa
      361 atctttactt attggtttca aaacccattg gttaagcctt ttaaactcat ggtagttatt
      421 ttcaagcatt aacatgaact taaattcatc aaggctaatc tctatatttg ccttgtgagt
      481 tttcttttgt gttagttctt ttaataacca ctcataaatc ctcatagagt atttgttttc
      541 aaaagactta acatgttcca gattatattt tatgaatttt tttaactgga aaagataagg
      601 caatatctct tcactaaaaa ctaattctaa tttttcgctt gagaacttgg catagtttgt
      661 ccactggaaa atctcaaagc ctttaaccaa aggattcctg atttccacag ttctcgtcat
      721 cagctctctg gttgctttag ctaatacacc ataagcattt tccctactga tgttcatcat
      781 ctgagcgtat tggttataag tgaacgatac cgtccgttct ttccttgtag ggttttcaat
      841 cgtggggttg agtagtgcca cacagcataa aattagcttg gtttcatgct ccgttaagtc
      901 atagcgacta atcgctagtt catttgcttt gaaaacaact aattcagaca tacatctcaa
      961 ttggtctagg taattttaat cactatacca attgagatgg gctagtcaat gataattact
     1021 agtccttttc ctttgagttg tgggtatctg taaattctgc tagacctttg ctggaaaact
     1081 tgtaaattct gctagaccct ctgtaaattc cgctagacct ttgtgtgttt tttttgttta
     1141 tattcaagtg gttataattt atagaataaa gaaagaataa aaaaagataa aaagaataga
     1201 tcccagccct gtgtataact cactacttta gtcagttccg cagtattaca aaaggatgtc
     1261 gcaaacgctg tttgctcctc tacaaaacag accttaaaac cctaaaggct taagtagcac
     1321 cctcgcaagc tcgggcaaat cgctgaatat tccttttgtc tccgaccatc aggcacctga
     1381 gtcgctgtct ttttcgtgac attcagttcg ctgcgctcac ggctctggca gtgaatgggg
     1441 gtaaatggca ctacaggcgc cttttatgga ttcatgcaag gaaactaccc ataatacaag
     1501 aaaagcccgt cacgggcttc tcagggcgtt ttatggcggg tctgctatgt ggtgctatct
     1561 gactttttgc tgttcagcag ttcctgccct ctgattttcc agtctgacca cttcggatta
     1621 tcccgtgaca ggtcattcag actggctaat gcacccagta aggcagcggt atcatcaaca
     1681 ggcttacccg tcgtacggtc aagaggacat ccgggcaaat gcaacgaaag gctcagtcga
     1741 aagactgggc ctttcgttgc gctgcggaga cttagggacc ctaatacaag aaagtttgtt
     1801 cattaggcac cccgggcttt acacgtaatg cttccggcgc gtatgttgtg tcgaccgacc
     1861 tgcaggtgca gtaaggagga aaaaaaaatg aaatttggaa actttttgct tacataccaa
     1921 cctccccaat tttcccaaac agaggtaatg aaacgtttgg ttaaattagg tcgcatctct
     1981 gaggagtgtg gttttgatac cgtatggtta ctggagcatc atttcacgga gtttggtttg
     2041 cttggtaacc cttatgtcgc tgctgcatat ttacttggcg cgactaaaaa attgaatgta
     2101 ggaactgccg ctattgttct tcccacagcc catccagtac gccaacttga agatgtgaat
     2161 ttattggatc aaatgtcaaa aggacgattt cggtttggta tttgccgagg gctttacaac
     2221 aaggactttc gcgtattcgg cacagatatg aataacagtc gcgccttagc ggaatgctgg
     2281 tacgggctga taaagaatgg catgacagag ggatatatgg aagctgataa tgaacatatc
     2341 aagttccata aggtaaaagt aaaccccgcg gcgtatagca gaggtggcgc accggtttat
     2401 gtggtggctg aatcagcttc gacgactgag tgggctgctc aatttggcct accgatgata
     2461 ttaagttgga ttataaatac taacgaaaag aaagcacaac ttgagcttta taatgaagtg
     2521 gctcaagaat atgggcacga tattcataat atcgaccatt gcttatcata tataacatct
     2581 gtagatcatg actcaattaa agcgaaagag atttgccgga aatttctggg gcattggtat
     2641 gattcttatg tgaatgctac gactattttt gatgattcag accaaacaag aggttatgat
     2701 ttcaataaag ggcagtggcg tgactttgta ttaaaaggac ataaagatac taatcgccgt
     2761 attgattaca gttacgaaat caatcccgtg ggaacgccgc aggaatgtat tgacataatt
     2821 caaaaagaca ttgatgctac aggaatatca aatatttgtt gtggatttga agctaatgga
     2881 acagtagacg aaattattgc ttccatgaag ctcttccagt ctgatgtcat gccatttctt
     2941 aaagaaaaac aacgttcgct attatattat ggcggtggcg gtagcggcgg tggcggtagc
     3001 ggcggtggcg gtagcggcgg tggcggtagc aaatttggat tgttcttcct taacttcatc
     3061 aattcaacaa ctgttcaaga acagagtata gttcgcatgc aggaaataac ggagtatgtt
     3121 gataagttga attttgaaca gattttagtg tatgaaaatc atttttcaga taatggtgtt
     3181 gtcggcgctc ctctgactgt ttctggtttt ctgctcggtt taacagagaa aattaaaatt
     3241 ggttcattaa atcacatcat tacaactcat catcctgtcc gcatagcgga ggaagcttgc
     3301 ttattggatc agttaagtga agggagattt attttagggt ttagtgattg cgaaaaaaaa
     3361 gatgaaatgc atttttttaa tcgcccggtt gaatatcaac agcaactatt tgaagagtgt
     3421 tatgaaatca ttaacgatgc tttaacaaca ggctattgta atccagataa cgatttttat
     3481 agcttcccta aaatatctgt aaatccccat gcttatacgc caggcggacc tcggaaatat
     3541 gtaacagcaa ccagtcatca tattgttgag tgggcggcca aaaaaggtat tcctctcatc
     3601 tttaagtggg atgattctaa tgatgttaga tatgaatatg ctgaaagata taaagccgtt
     3661 gcggataaat atgacgttga cctatcagag atagaccatc agttaatgat attagttaac
     3721 tataacgaag atagtaataa agctaaacaa gagacgcgtg catttattag tgattatgtt
     3781 cttgaaatgc accctaatga aaatttcgaa aataaacttg aagaaataat tgcagaaaac
     3841 gctgtcggaa attatacgga gtgtataact gcggctaagt tggcaattga aaagtgtggt
     3901 gcgaaaagtg tattgctgtc ctttgaacca atgaatgatt tgatgagcca aaaaaatgta
     3961 atcaatattg ttgatgataa tattaagaag taccacacgg aatataccta aacggagcca
     4021 atgtacgcaa aaaaccccgc ttcagcgggg ttttttcgcc aaaaaaaacc ccgccctgtc
     4081 aggggcgggg ttttttttta gggccctact gactgttatg tgaggctgtt gtccacgccc
     4141 agctctgggc tggagccacc cacaaagtcc tccagatgca gctcgcgagt taacagggtt
     4201 tcctgaatcg cacgcagcag tattgtgtta ctacggagct gctcgatctc gtcggtggtg
     4261 gcgggcacgt ggttccgtat gaagtgcgcg cgcacctcgg tgcggtcgtc gcgcgcggcg
     4321 ccgctgctgt ccggcgcgcg cagcaccgcg tcgatgttgc acgtcaggct gaacaccgac
     4381 gagaacgtgt ccgcgatgaa agcacggttg gagtcaacga catccagcgt gttgttgaac
     4441 aagaagtaga ctctgttgag atgagtgatc agatagatct tcacgtcagt acgggcttct
     4501 gcaccacggg tgtctgtagc cacgacatcg aactcgaaca tgccgaccat agtggcgctc
     4561 gggttgaagt tgagggacag gacgccgctg ctcgggtgca gcgtgaaggc ggactcacgc
     4621 acgccctcca gcgaggggtc caccaccatc gtgtccaggt ccatcgtgta cgttatgtct
     4681 aggccttcgg tatgtgtcgc atgaacagtg agtagttccc tgttggatgt atccgtctcg
     4741 tagatgccgg cggtgtagat ctcacgctcg aatactggcc gcttgtcatc gtcgtccttg
     4801 tagtctacga attttaccct cgcttccacg acaacaccga taatcttgca gtttccgttg
     4861 ataggagtca taggccatga aggattcagg cctttcaggt acttctgacc gccatctatg
     4921 accagtttct tgaatgttgc ttcgttcgcg tcagtcagtt tggctacaac aaggcttcca
     4981 ttcactggct cccgtccagt atctactaac accatatgac cttcagggat gctttgacct
     5041 acaggtgagg tcatggaatc accttcaacc ttcagccaga atccattgcc taataagtta
     5101 acgtcactgt cataccattc atcaatgtcc ttgatatcgt agggttcaca agcttcacac
     5161 cacgaacgag ctctaaccat gctaatcaat ggatatttcc ctttgggctc aacgtgccca
     5221 acaaatctaa cattcgaatc agaggtgcca ttgagcagcc agtcaacact tacgccaaga
     5281 gctgacgcaa gttctggtaa aaagcgtggt cgcttagttt taccgttttc gagctgctct
     5341 atagactgct gggtagtccc caccttttga gcaagttcag cctggttaag tccaagctga
     5401 attcttttgc tttttaccct gctgctgata ctcatttttt tttcctcctt actctagtaa
     5461 aagttaaaca aaattatttg tagagggaaa ccgttgtggt ctcccttctc tatcactgat
     5521 agggagtggt aaaataactc tatcaatgat agagtgtcaa tctctatcac tgatagggac
     5581 agcaaccact catagaccta gggcagcaga tagggacgac gtggtgttag ctgtgctcta
     5641 gaatcctcag cgagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt
     5701 aatttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt
     5761 cgcaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa
     5821 acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct
     5881 ggcagttccc tactctcgca tggggagacc ccacactacc atcggcgcta cggcgtttca
     5941 cttctgagtt cggcatgggg tcaggtggga ccaccgcgct actgccgcca ggcaaattct
     6001 gttttatcag accgcttctg cgttctgatt taatctgtat caggctgaaa atcttctctc
     6061 atccgccaaa acagccatgc ccatggcgtt taccaatgct taatcagtga ggcacctatc
     6121 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6181 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6241 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6301 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6361 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6421 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6481 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6541 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6601 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6661 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6721 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6781 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6841 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6901 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6961 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7021 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7081 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     7141 cccttaggcc aaataggccg t
//
