LOCUS       sequence-199782-        4265 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 4265)
  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 4265)
  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..4265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     149..167
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             226..939
                     /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(256..284)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     879..899
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      980..1066
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1604..2554)
                     /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      2602..2824
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3316..3410)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3441..4235)
                     /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(3552..3571)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4162..4181
                     /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 cctcgatgtc gagagatcct ctagatttaa gaaggagata tacatatgag taaaggagaa
      241 gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac
      301 aaattttctg tcagtggaga gggtgaaggt gatgcaacat acggaaaact tacccttaaa
      361 tttatttgca ctactggaaa actacctgtt ccatggccaa cacttgtcac tactttcgcg
      421 tatggtcttc aatgctttgc gagataccca gatcatatga aacagcatga ctttttcaag
      481 agtgccatgc ccgaaggtta tgtacaggaa agaactatat ttttcaaaga tgacgggaac
      541 tacaagacac gtgctgaagt caagtttgaa ggtgataccc ttgttaatag aatcgagtta
      601 aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga atacaactat
      661 aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa agttaacttc
      721 aaaattagac acaacattga agatggaagc gttcaactag cagaccatta tcaacaaaat
      781 actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc cacacaatct
      841 gccctttcga aagatcccaa cgaaaagaga gaccacatgg tccttcttga gtttgtaaca
      901 gctgctggga ttacacatgg catggatgaa ctatacaaat aaatgtccag acctgcaggc
      961 atgcaagctc tagaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     1021 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgccctagac
     1081 ctagggtacg ggttttgctg cccgcaaacg ggctgttctg gtgttgctag tttgttatca
     1141 gaatcgcaga tccggcttca gccggtttgc cggctgaaag cgctatttct tccagaattg
     1201 ccatgatttt ttccccacgg gaggcgtcac tggctcccgt gttgtcggca gctttgattc
     1261 gataagcagc atcgcctgtt tcaggctgtc tatgtgtgac tgttgagctg taacaagttg
     1321 tctcaggtgt tcaatttcat gttctagttg ctttgtttta ctggtttcac ctgttctatt
     1381 aggtgttaca tgctgttcat ctgttacatt gtcgatctgt tcatggtgaa cagctttgaa
     1441 tgcaccaaaa actcgtaaaa gctctgatgt atctatcttt tttacaccgt tttcatctgt
     1501 gcatatggac agttttccct ttgatatgta acggtgaaca gttgttctac ttttgtttgt
     1561 tagtcttgat gcttcactga tagatacaag agccataaga acctcagatc cttccgtatt
     1621 tagccagtat gttctctagt gtggttcgtt gtttttgcgt gagccatgag aacgaaccat
     1681 tgagatcata cttactttgc atgtcactca aaaattttgc ctcaaaactg gtgagctgaa
     1741 tttttgcagt taaagcatcg tgtagtgttt ttcttagtcc gttatgtagg taggaatctg
     1801 atgtaatggt tgttggtatt ttgtcaccat tcatttttat ctggttgttc tcaagttcgg
     1861 ttacgagatc catttgtcta tctagttcaa cttggaaaat caacgtatca gtcgggcggc
     1921 ctcgcttatc aaccaccaat ttcatattgc tgtaagtgtt taaatcttta cttattggtt
     1981 tcaaaaccca ttggttaagc cttttaaact catggtagtt attttcaagc attaacatga
     2041 acttaaattc atcaaggcta atctctatat ttgccttgtg agttttcttt tgtgttagtt
     2101 cttttaataa ccactcataa atcctcatag agtatttgtt ttcaaaagac ttaacatgtt
     2161 ccagattata ttttatgaat ttttttaact ggaaaagata aggcaatatc tcttcactaa
     2221 aaactaattc taatttttcg cttgagaact tggcatagtt tgtccactgg aaaatctcaa
     2281 agcctttaac caaaggattc ctgatttcca cagttctcgt catcagctct ctggttgctt
     2341 tagctaatac accataagca ttttccctac tgatgttcat catctgagcg tattggttat
     2401 aagtgaacga taccgtccgt tctttccttg tagggttttc aatcgtgggg ttgagtagtg
     2461 ccacacagca taaaattagc ttggtttcat gctccgttaa gtcatagcga ctaatcgcta
     2521 gttcatttgc tttgaaaaca actaattcag acatacatct caattggtct aggtgatttt
     2581 aatcactata ccaattgaga tgggctagtc aatgataatt actagtcctt ttcctttgag
     2641 ttgtgggtat ctgtaaattc tgctagacct ttgctggaaa acttgtaaat tctgctagac
     2701 cctctgtaaa ttccgctaga cctttgtgtg ttttttttgt ttatattcaa gtggttataa
     2761 tttatagaat aaagaaagaa taaaaaaaga taaaaagaat agatcccagc cctgtgtata
     2821 actcactact ttagtcagtt ccgcagtatt acaaaaggat gtcgcaaacg ctgtttgctc
     2881 ctctacaaaa cagaccttaa aaccctaaag gcttaagtag cgccgtcgca agctccggca
     2941 aatcgctgaa tattcctttt gtctccgacc atcaggcacc tgagtcgctg tctttttcgt
     3001 cacattcagt tcgctgcgct cacggctctg gcagtgaatg ggggtaaatg gcactacagg
     3061 cgccttttat ggattcatgc aaggaaacta cccataatac aagaaaagcc cgtcacgggc
     3121 ttctcagggc gttttatggc gggtctgcta tgtggtgcta tctgactttt tgctgttcag
     3181 cagttcctgc cctctgattt tccagtctga ccacttcgga ttatcccgtg acaggtcatt
     3241 cagactggct aatgcaccca gtaaggcagc ggtatcatca acaggcttac ccgtcttact
     3301 gtccctagtg cttggattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa
     3361 caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct
     3421 ctcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc
     3481 tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca
     3541 agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc
     3601 agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag
     3661 caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg
     3721 gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca
     3781 agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat
     3841 gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact
     3901 ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc
     3961 agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc
     4021 gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg
     4081 tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca
     4141 gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc
     4201 ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct
     4261 tgatc
//
