LOCUS       sequence-210134-        4277 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP with no promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4277)
  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 4277)
  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 theta GFP
FEATURES             Location/Qualifiers
     source          1..4277
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(170..264)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(295..1089)
                     /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(406..425)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1016..1035
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1268..1286
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             1357..2070
                     /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(1387..1415)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2010..2030
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2111..2197
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2735..3685)
                     /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      3733..3955
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 gctatgtggt gctatctgac tttttgctgt tcagcagttc ctgccctctg attttccagt
       61 ctgaccactt cggattatcc cgtgacaggt cattcagact ggctaatgca cccagtaagg
      121 cagcggtatc atcaacaggc ttacccgtct tactgtccct agtgcttgga ttctcaccaa
      181 taaaaaacgc ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt tctgaggtca
      241 ttactggatc tatcaacagg agtccaagcg agctctcgaa ccccagagtc ccgctcagaa
      301 gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta
      361 aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc
      421 caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga
      481 aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag
      541 atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc
      601 ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc
      661 tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg
      721 cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga
      781 caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac
      841 aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc
      901 ctcgtcctgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg
      961 cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca
     1021 gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg
     1081 ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gatcttgatc ccctgcgcca
     1141 tcagatcctt ggcggcaaga aagccatcca gtttactttg cagggcttcc caaccttacc
     1201 agagggcgcc ccagctggca attccgacgt ctaagaaacc attattatca tgacattaac
     1261 ctataaaaat aggcgtatca cgaggccctt tcgtcttcac ctcgatggat ccgaattcct
     1321 cgagagatcc tctagattta agaaggagat atacatatga gtaaaggaga agaacttttc
     1381 actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct
     1441 gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc
     1501 actactggaa aactacctgt tccatggcca acacttgtca ctactttcgc gtatggtctt
     1561 caatgctttg cgagataccc agatcatatg aaacagcatg actttttcaa gagtgccatg
     1621 cccgaaggtt atgtacagga aagaactata tttttcaaag atgacgggaa ctacaagaca
     1681 cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt
     1741 gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacta taactcacac
     1801 aatgtataca tcatggcaga caaacaaaag aatggaatca aagttaactt caaaattaga
     1861 cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa tactccaatt
     1921 ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc tgccctttcg
     1981 aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg
     2041 attacacatg gcatggatga actatacaaa taaatgtcca gacctgcagg catgcaagct
     2101 ctagaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     2161 ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgccctaga cctagggtac
     2221 gggttttgct gcccgcaaac gggctgttct ggtgttgcta gtttgttatc agaatcgcag
     2281 atccggcttc agccggtttg ccggctgaaa gcgctatttc ttccagaatt gccatgattt
     2341 tttccccacg ggaggcgtca ctggctcccg tgttgtcggc agctttgatt cgataagcag
     2401 catcgcctgt ttcaggctgt ctatgtgtga ctgttgagct gtaacaagtt gtctcaggtg
     2461 ttcaatttca tgttctagtt gctttgtttt actggtttca cctgttctat taggtgttac
     2521 atgctgttca tctgttacat tgtcgatctg ttcatggtga acagctttga atgcaccaaa
     2581 aactcgtaaa agctctgatg tatctatctt ttttacaccg ttttcatctg tgcatatgga
     2641 cagttttccc tttgatatgt aacggtgaac agttgttcta cttttgtttg ttagtcttga
     2701 tgcttcactg atagatacaa gagccataag aacctcagat ccttccgtat ttagccagta
     2761 tgttctctag tgtggttcgt tgtttttgcg tgagccatga gaacgaacca ttgagatcat
     2821 acttactttg catgtcactc aaaaattttg cctcaaaact ggtgagctga atttttgcag
     2881 ttaaagcatc gtgtagtgtt tttcttagtc cgttatgtag gtaggaatct gatgtaatgg
     2941 ttgttggtat tttgtcacca ttcattttta tctggttgtt ctcaagttcg gttacgagat
     3001 ccatttgtct atctagttca acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat
     3061 caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc
     3121 attggttaag ccttttaaac tcatggtagt tattttcaag cattaacatg aacttaaatt
     3181 catcaaggct aatctctata tttgccttgt gagttttctt ttgtgttagt tcttttaata
     3241 accactcata aatcctcata gagtatttgt tttcaaaaga cttaacatgt tccagattat
     3301 attttatgaa tttttttaac tggaaaagat aaggcaatat ctcttcacta aaaactaatt
     3361 ctaatttttc gcttgagaac ttggcatagt ttgtccactg gaaaatctca aagcctttaa
     3421 ccaaaggatt cctgatttcc acagttctcg tcatcagctc tctggttgct ttagctaata
     3481 caccataagc attttcccta ctgatgttca tcatctgagc gtattggtta taagtgaacg
     3541 ataccgtccg ttctttcctt gtagggtttt caatcgtggg gttgagtagt gccacacagc
     3601 ataaaattag cttggtttca tgctccgtta agtcatagcg actaatcgct agttcatttg
     3661 ctttgaaaac aactaattca gacatacatc tcaattggtc taggtgattt taatcactat
     3721 accaattgag atgggctagt caatgataat tactagtcct tttcctttga gttgtgggta
     3781 tctgtaaatt ctgctagacc tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa
     3841 attccgctag acctttgtgt gttttttttg tttatattca agtggttata atttatagaa
     3901 taaagaaaga ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat aactcactac
     3961 tttagtcagt tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa
     4021 acagacctta aaaccctaaa ggcttaagta gcaccctcgc aagctcgggc aaatcgctga
     4081 atattccttt tgtctccgac catcaggcac ctgagtcgct gtctttttcg tgacattcag
     4141 ttcgctgcgc tcacggctct ggcagtgaat gggggtaaat ggcactacag gcgcctttta
     4201 tggattcatg caaggaaact acccataata caagaaaagc ccgtcacggg cttctcaggg
     4261 cgttttatgg cgggtct
//
