LOCUS       sequence-199785-        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"
     primer_bind     149..167
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             557..1270
                     /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(587..615)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1210..1230
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1311..1397
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1935..2885)
                     /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      2933..3155
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3647..3741)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3772..4566)
                     /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(3883..3902)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4493..4512
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 agatcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt
       61 gcagggcttc ccaaccttac cagagggcgc cccagctggc aattccgacg tctaagaaac
      121 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca
      181 cctcgatgga tccccgcggt ttgactggtc aaatttcgaa cgacatcatt ctcattgact
      241 atactgattt cgtcagactt acggttaagc accccagcca gatggcctgg tgatggcggg
      301 atcgttgtat atttcttgac accttttcgg catcgcccta aaattcggcg tcctcatatt
      361 gtgtgaggac gttttattac gtgtttacga agcaaaagct aaaaccagga gctatttaat
      421 ggcaacagtt aaccagctgg tacgcaaacc acgtgctcgc aaagttgcga aaagcaacgt
      481 gcctgcgctg gaagcatgcc cgcaaaaacg tggcgtatct cgagagatcc tctagattta
      541 agaaggagat atacatatga gtaaaggaga agaacttttc actggagttg tcccaattct
      601 tgttgaatta gatggtgatg ttaatgggca caaattttct gtcagtggag agggtgaagg
      661 tgatgcaaca tacggaaaac ttacccttaa atttatttgc actactggaa aactacctgt
      721 tccatggcca acacttgtca ctactttcgc gtatggtctt caatgctttg cgagataccc
      781 agatcatatg aaacagcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga
      841 aagaactata tttttcaaag atgacgggaa ctacaagaca cgtgctgaag tcaagtttga
      901 aggtgatacc cttgttaata gaatcgagtt aaaaggtatt gattttaaag aagatggaaa
      961 cattcttgga cacaaattgg aatacaacta taactcacac aatgtataca tcatggcaga
     1021 caaacaaaag aatggaatca aagttaactt caaaattaga cacaacattg aagatggaag
     1081 cgttcaacta gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt
     1141 accagacaac cattacctgt ccacacaatc tgccctttcg aaagatccca acgaaaagag
     1201 agaccacatg gtccttcttg agtttgtaac agctgctggg attacacatg gcatggatga
     1261 actatacaaa taaatgtcca gacctgcagg catgcaagct ctagaggcat caaataaaac
     1321 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     1381 tcctgagtag gacaaatccg ccgccctaga cctagggtac gggttttgct gcccgcaaac
     1441 gggctgttct ggtgttgcta gtttgttatc agaatcgcag atccggcttc agccggtttg
     1501 ccggctgaaa gcgctatttc ttccagaatt gccatgattt tttccccacg ggaggcgtca
     1561 ctggctcccg tgttgtcggc agctttgatt cgataagcag catcgcctgt ttcaggctgt
     1621 ctatgtgtga ctgttgagct gtaacaagtt gtctcaggtg ttcaatttca tgttctagtt
     1681 gctttgtttt actggtttca cctgttctat taggtgttac atgctgttca tctgttacat
     1741 tgtcgatctg ttcatggtga acagctttga atgcaccaaa aactcgtaaa agctctgatg
     1801 tatctatctt ttttacaccg ttttcatctg tgcatatgga cagttttccc tttgatatgt
     1861 aacggtgaac agttgttcta cttttgtttg ttagtcttga tgcttcactg atagatacaa
     1921 gagccataag aacctcagat ccttccgtat ttagccagta tgttctctag tgtggttcgt
     1981 tgtttttgcg tgagccatga gaacgaacca ttgagatcat acttactttg catgtcactc
     2041 aaaaattttg cctcaaaact ggtgagctga atttttgcag ttaaagcatc gtgtagtgtt
     2101 tttcttagtc cgttatgtag gtaggaatct gatgtaatgg ttgttggtat tttgtcacca
     2161 ttcattttta tctggttgtt ctcaagttcg gttacgagat ccatttgtct atctagttca
     2221 acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat caaccaccaa tttcatattg
     2281 ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc attggttaag ccttttaaac
     2341 tcatggtagt tattttcaag cattaacatg aacttaaatt catcaaggct aatctctata
     2401 tttgccttgt gagttttctt ttgtgttagt tcttttaata accactcata aatcctcata
     2461 gagtatttgt tttcaaaaga cttaacatgt tccagattat attttatgaa tttttttaac
     2521 tggaaaagat aaggcaatat ctcttcacta aaaactaatt ctaatttttc gcttgagaac
     2581 ttggcatagt ttgtccactg gaaaatctca aagcctttaa ccaaaggatt cctgatttcc
     2641 acagttctcg tcatcagctc tctggttgct ttagctaata caccataagc attttcccta
     2701 ctgatgttca tcatctgagc gtattggtta taagtgaacg ataccgtccg ttctttcctt
     2761 gtagggtttt caatcgtggg gttgagtagt gccacacagc ataaaattag cttggtttca
     2821 tgctccgtta agtcatagcg actaatcgct agttcatttg ctttgaaaac aactaattca
     2881 gacatacatc tcaattggtc taggtgattt taatcactat accaattgag atgggctagt
     2941 caatgataat tactagtcct tttcctttga gttgtgggta tctgtaaatt ctgctagacc
     3001 tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa attccgctag acctttgtgt
     3061 gttttttttg tttatattca agtggttata atttatagaa taaagaaaga ataaaaaaag
     3121 ataaaaagaa tagatcccag ccctgtgtat aactcactac tttagtcagt tccgcagtat
     3181 tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa acagacctta aaaccctaaa
     3241 ggcttaagta gcgccgtcgc aagctccggc aaatcgctga atattccttt tgtctccgac
     3301 catcaggcac ctgagtcgct gtctttttcg tcacattcag ttcgctgcgc tcacggctct
     3361 ggcagtgaat gggggtaaat ggcactacag gcgcctttta tggattcatg caaggaaact
     3421 acccataata caagaaaagc ccgtcacggg cttctcaggg cgttttatgg cgggtctgct
     3481 atgtggtgct atctgacttt ttgctgttca gcagttcctg ccctctgatt ttccagtctg
     3541 accacttcgg attatcccgt gacaggtcat tcagactggc taatgcaccc agtaaggcag
     3601 cggtatcatc aacaggctta cccgtcttac tgtccctagt gcttggattc tcaccaataa
     3661 aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta
     3721 ctggatctat caacaggagt ccaagcgagc tctcgaaccc cagagtcccg ctcagaagaa
     3781 ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag
     3841 cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa
     3901 cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa
     3961 gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc
     4021 ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg
     4081 atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg
     4141 ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag
     4201 ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag
     4261 gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac
     4321 gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc
     4381 gtcctgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc
     4441 ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc
     4501 atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc
     4561 aatcatgcga aacgatcctc atcctgtctc ttgatc
//
