LOCUS       sequence-476950-        6766 bp    DNA     circular SYN 24-JUN-2025
DEFINITION  Directed Evolution of Protein-Protein Interactions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6766)
  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 6766)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 24, 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: pAB064d
FEATURES             Location/Qualifiers
     source          1..6766
                     /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"
     promoter        join(1818..1823,1824..1841,1842..1848)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     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(4419..4442)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      complement(5274..5301)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(5393..5479)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5632..5649
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(join(5695..6486,6487..6555))
                     /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     6318..6337
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6556..6660)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6728..6746
                     /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 tcttactgtc aagaggacat ccggtcaaat aaaacgaaag gctcagtcga
     1741 aagactgggc ctttcgtttt gctgaggaga cttagggacc ctaatacaag aaagtttgtt
     1801 cattaggcac cccgggcttt acactttatg cttccggctc 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 gactgttagc gtttcggcgc cggataatgc
     4141 agcgtggtga tcagaccatc cgcaaccgtg ctatggtgat gcaccagttc tgccagggta
     4201 ttgaagcgag attcactaga aacgtacagt ttgccatcgc tggcggtgtt gatacggtag
     4261 tgatacacgc gaccttcata acgcagagaa atgctgcgct ggcccgggct actttcactt
     4321 tcacgaacca gaaaagaacc attaatgcca gagctcagca ggtattcggc cgcgttacgg
     4381 ctcacagggc catgatacca gctgtgtttt tccagactct tgtcatcgtc gtccttgtag
     4441 tctacgaatt ttaccctcgc ttccacgaca acaccgataa tcttgcagtt tccgttgata
     4501 ggagtcatag gccatgaagg attcaggcct ttcaggtact tctgaccgcc atctatgacc
     4561 agtttcttga atgttgcttc gttcgcgtca gtcagtttgg ctacaacaag gcttccattc
     4621 actggctccc gtccagtatc tactaacacc atatgacctt cagggatgct ttgacctaca
     4681 ggtgaggtca tggaatcacc ttcaaccttc agccagaatc cattgcctaa taagttaacg
     4741 tcactgtcat accattcatc aatgtccttg atatcgtagg gttcacaagc ttcacaccac
     4801 gaacgagctc taaccatgct aatcaatgga tatttccctt tgggctcaac gtgcccaaca
     4861 aatctaacat tcgaatcaga ggtgccattg agcagccagt caacacttac gccaagagct
     4921 gacgcaagtt ctggtaaaaa gcgtggtcgc ttagttttac cgttttcgag ctgctctata
     4981 gactgctggg tagtccccac cttttgagca agttcagcct ggttaagtcc aagctgaatt
     5041 cttttgcttt ttaccctgct gctgatactc attttttttt cctccttact ctagtaaaag
     5101 ttaaacaaaa ttatttgtag agggaaaccg ttgtggtctc cctgaatata gataccgagc
     5161 cttatgcatg cccgtaaagt tatccagcaa ccactcatag acctagggca gcagataggg
     5221 acgacgtggt gttagctgtg ctctagaatc ctcagcgagt ttgtagaaac gcaaaaaggc
     5281 catccgtcag gatggccttc tgcttaattt gatgcctggc agtttatggc gggcgtcctg
     5341 cccgccaccc tccgggccgt tgcttcgcaa cgttcaaatc cgctcccggc ggatttgtcc
     5401 tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact
     5461 gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcatgggg agaccccaca
     5521 ctaccatcgg cgctacggcg tttcacttct gagttcggca tggggtcagg tgggaccacc
     5581 gcgctactgc cgccaggcaa attctgtttt atcagaccgc ttctgcgttc tgatttaatc
     5641 tgtatcaggc tgaaaatctt ctctcatccg ccaaaacagc catgcccatg gcgtttacca
     5701 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5761 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5821 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5881 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5941 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6001 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6061 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6121 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6181 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6241 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6301 cccagcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6361 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6421 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6481 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6541 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6601 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6661 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     6721 ctataaaaat aggcgtatca cgaggccctt aggccaaata ggccgt
//
