LOCUS       sequence-210135-        4596 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP under PrpsL.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4596)
  AUTHORS   Stirling F, Bitzan L, O'Keefe S, Redfield E, Oliver JWK, Way J, 
            Silver PA
  TITLE     Rational Design of Evolutionarily Stable Microbial Kill Switches.
  JOURNAL   Mol Cell. 2017 Nov 16;68(4):686-697.e3. doi: 
            10.1016/j.molcel.2017.10.033.
  PUBMED    29149596
REFERENCE   2  (bases 1 to 4596)
  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: 
            "10.1016/j.molcel.2017.10"; journalName: "Mol Cell"; date: 
            "2017-11-16- 16"; volume: "68"; issue: "4"; pages: "686-697.e3"
COMMENT     Sequence Label: pUA66 PrpsL GFP
FEATURES             Location/Qualifiers
     source          1..4596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(171..265)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(296..1090)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(407..426)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1017..1036
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1269..1287
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             1677..2390
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1707..1735)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2330..2350
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2431..2517
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3055..4005)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      4053..4275
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 tgctatgtgg tgctatctga ctttttgctg ttcagcagtt cctgccctct gattttccag
       61 tctgaccact tcggattatc ccgtgacagg tcattcagac tggctaatgc acccagtaag
      121 gcagcggtat catcaacagg cttacccgtc ttactgtccc tagtgcttgg attctcacca
      181 ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc
      241 attactggat ctatcaacag gagtccaagc gagctctcga accccagagt cccgctcaga
      301 agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt
      361 aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag
      421 ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag
      481 aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg gtcacgacga
      541 gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc
      601 cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc cgagtacgtg
      661 ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga tcaagcgtat
      721 gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca aggtgagatg
      781 acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc gcttcagtga
      841 caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat agccgcgctg
      901 cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa agaaccgggc
      961 gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc tgttgtgccc
     1021 agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc aatccatctt
     1081 gttcaatcat gcgaaacgat cctcatcctg tctcttgatc agatcttgat cccctgcgcc
     1141 atcagatcct tggcggcaag aaagccatcc agtttacttt gcagggcttc ccaaccttac
     1201 cagagggcgc cccagctggc aattccgacg tctaagaaac cattattatc atgacattaa
     1261 cctataaaaa taggcgtatc acgaggccct ttcgtcttca cctcgatgga tccccgcggt
     1321 ttgactggtc aaatttcgaa cgacatcatt ctcattgact atactgattt cgtcagactt
     1381 acggttaagc accccagcca gatggcctgg tgatggcggg atcgttgtat atttcttgac
     1441 accttttcgg catcgcccta aaattcggcg tcctcatatt gtgtgaggac gttttattac
     1501 gtgtttacga agcaaaagct aaaaccagga gctatttaat ggcaacagtt aaccagctgg
     1561 tacgcaaacc acgtgctcgc aaagttgcga aaagcaacgt gcctgcgctg gaagcatgcc
     1621 cgcaaaaacg tggcgtatct cgagagatcc tctagattta agaaggagat atacatatga
     1681 gtaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg
     1741 ttaatgggca caaattttct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac
     1801 ttacccttaa atttatttgc actactggaa aactacctgt tccatggcca acacttgtca
     1861 ctactttcgc gtatggtctt caatgctttg cgagataccc agatcatatg aaacagcatg
     1921 actttttcaa gagtgccatg cccgaaggtt atgtacagga aagaactata tttttcaaag
     1981 atgacgggaa ctacaagaca cgtgctgaag tcaagtttga aggtgatacc cttgttaata
     2041 gaatcgagtt aaaaggtatt gattttaaag aagatggaaa cattcttgga cacaaattgg
     2101 aatacaacta taactcacac aatgtataca tcatggcaga caaacaaaag aatggaatca
     2161 aagttaactt caaaattaga cacaacattg aagatggaag cgttcaacta gcagaccatt
     2221 atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt
     2281 ccacacaatc tgccctttcg aaagatccca acgaaaagag agaccacatg gtccttcttg
     2341 agtttgtaac agctgctggg attacacatg gcatggatga actatacaaa taaatgtcca
     2401 gacctgcagg catgcaagct ctagaggcat caaataaaac gaaaggctca gtcgaaagac
     2461 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
     2521 ccgccctaga cctagggtac gggttttgct gcccgcaaac gggctgttct ggtgttgcta
     2581 gtttgttatc agaatcgcag atccggcttc agccggtttg ccggctgaaa gcgctatttc
     2641 ttccagaatt gccatgattt tttccccacg ggaggcgtca ctggctcccg tgttgtcggc
     2701 agctttgatt cgataagcag catcgcctgt ttcaggctgt ctatgtgtga ctgttgagct
     2761 gtaacaagtt gtctcaggtg ttcaatttca tgttctagtt gctttgtttt actggtttca
     2821 cctgttctat taggtgttac atgctgttca tctgttacat tgtcgatctg ttcatggtga
     2881 acagctttga atgcaccaaa aactcgtaaa agctctgatg tatctatctt ttttacaccg
     2941 ttttcatctg tgcatatgga cagttttccc tttgatatgt aacggtgaac agttgttcta
     3001 cttttgtttg ttagtcttga tgcttcactg atagatacaa gagccataag aacctcagat
     3061 ccttccgtat ttagccagta tgttctctag tgtggttcgt tgtttttgcg tgagccatga
     3121 gaacgaacca ttgagatcat acttactttg catgtcactc aaaaattttg cctcaaaact
     3181 ggtgagctga atttttgcag ttaaagcatc gtgtagtgtt tttcttagtc cgttatgtag
     3241 gtaggaatct gatgtaatgg ttgttggtat tttgtcacca ttcattttta tctggttgtt
     3301 ctcaagttcg gttacgagat ccatttgtct atctagttca acttggaaaa tcaacgtatc
     3361 agtcgggcgg cctcgcttat caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt
     3421 acttattggt ttcaaaaccc attggttaag ccttttaaac tcatggtagt tattttcaag
     3481 cattaacatg aacttaaatt catcaaggct aatctctata tttgccttgt gagttttctt
     3541 ttgtgttagt tcttttaata accactcata aatcctcata gagtatttgt tttcaaaaga
     3601 cttaacatgt tccagattat attttatgaa tttttttaac tggaaaagat aaggcaatat
     3661 ctcttcacta aaaactaatt ctaatttttc gcttgagaac ttggcatagt ttgtccactg
     3721 gaaaatctca aagcctttaa ccaaaggatt cctgatttcc acagttctcg tcatcagctc
     3781 tctggttgct ttagctaata caccataagc attttcccta ctgatgttca tcatctgagc
     3841 gtattggtta taagtgaacg ataccgtccg ttctttcctt gtagggtttt caatcgtggg
     3901 gttgagtagt gccacacagc ataaaattag cttggtttca tgctccgtta agtcatagcg
     3961 actaatcgct agttcatttg ctttgaaaac aactaattca gacatacatc tcaattggtc
     4021 taggtgattt taatcactat accaattgag atgggctagt caatgataat tactagtcct
     4081 tttcctttga gttgtgggta tctgtaaatt ctgctagacc tttgctggaa aacttgtaaa
     4141 ttctgctaga ccctctgtaa attccgctag acctttgtgt gttttttttg tttatattca
     4201 agtggttata atttatagaa taaagaaaga ataaaaaaag ataaaaagaa tagatcccag
     4261 ccctgtgtat aactcactac tttagtcagt tccgcagtat tacaaaagga tgtcgcaaac
     4321 gctgtttgct cctctacaaa acagacctta aaaccctaaa ggcttaagta gcaccctcgc
     4381 aagctcgggc aaatcgctga atattccttt tgtctccgac catcaggcac ctgagtcgct
     4441 gtctttttcg tgacattcag ttcgctgcgc tcacggctct ggcagtgaat gggggtaaat
     4501 ggcactacag gcgcctttta tggattcatg caaggaaact acccataata caagaaaagc
     4561 ccgtcacggg cttctcaggg cgttttatgg cgggtc
//
