LOCUS       sequence-199780-        4494 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP under temperature sensitive promoter PcspA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4494)
  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 4494)
  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 PcspA GFP Linker
FEATURES             Location/Qualifiers
     source          1..4494
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     149..167
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     5'UTR           268..397
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             455..1168
                     /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(485..513)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1108..1128
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1209..1295
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1833..2783)
                     /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      2831..3053
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3545..3639)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3670..4464)
                     /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(3781..3800)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4391..4410
                     /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 cctcgatgat atgaaaaata attgttgcat cacccgccaa tgcgtggctt aatgcacatc
      241 aacggtttga cgtacagacc attaaagcag tgtagtaagg caagtccctt caagagttat
      301 cgttgatacc cctcgtagtg cacattcctt taacgcttca aaatctgtaa agcacgccat
      361 atcgccgaaa ggcacactta attattaaag gtaatacact atgtccggta aaatgactgg
      421 tatcgtaaaa tggttcaacg gtggaggtgg aagcatgagt aaaggagaag aacttttcac
      481 tggagttgtc ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt
      541 cagtggagag ggtgaaggtg atgcaacata cggaaaactt acccttaaat ttatttgcac
      601 tactggaaaa ctacctgttc catggccaac acttgtcact actttcgcgt atggtcttca
      661 atgctttgcg agatacccag atcatatgaa acagcatgac tttttcaaga gtgccatgcc
      721 cgaaggttat gtacaggaaa gaactatatt tttcaaagat gacgggaact acaagacacg
      781 tgctgaagtc aagtttgaag gtgataccct tgttaataga atcgagttaa aaggtattga
      841 ttttaaagaa gatggaaaca ttcttggaca caaattggaa tacaactata actcacacaa
      901 tgtatacatc atggcagaca aacaaaagaa tggaatcaaa gttaacttca aaattagaca
      961 caacattgaa gatggaagcg ttcaactagc agaccattat caacaaaata ctccaattgg
     1021 cgatggccct gtccttttac cagacaacca ttacctgtcc acacaatctg ccctttcgaa
     1081 agatcccaac gaaaagagag accacatggt ccttcttgag tttgtaacag ctgctgggat
     1141 tacccatggt atggatgaat tgtacaaata aatgtccaga cctgcaggca tgcaagctct
     1201 agaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     1261 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gccctagacc tagggtacgg
     1321 gttttgctgc ccgcaaacgg gctgttctgg tgttgctagt ttgttatcag aatcgcagat
     1381 ccggcttcag ccggtttgcc ggctgaaagc gctatttctt ccagaattgc catgattttt
     1441 tccccacggg aggcgtcact ggctcccgtg ttgtcggcag ctttgattcg ataagcagca
     1501 tcgcctgttt caggctgtct atgtgtgact gttgagctgt aacaagttgt ctcaggtgtt
     1561 caatttcatg ttctagttgc tttgttttac tggtttcacc tgttctatta ggtgttacat
     1621 gctgttcatc tgttacattg tcgatctgtt catggtgaac agctttgaat gcaccaaaaa
     1681 ctcgtaaaag ctctgatgta tctatctttt ttacaccgtt ttcatctgtg catatggaca
     1741 gttttccctt tgatatgtaa cggtgaacag ttgttctact tttgtttgtt agtcttgatg
     1801 cttcactgat agatacaaga gccataagaa cctcagatcc ttccgtattt agccagtatg
     1861 ttctctagtg tggttcgttg tttttgcgtg agccatgaga acgaaccatt gagatcatac
     1921 ttactttgca tgtcactcaa aaattttgcc tcaaaactgg tgagctgaat ttttgcagtt
     1981 aaagcatcgt gtagtgtttt tcttagtccg ttatgtaggt aggaatctga tgtaatggtt
     2041 gttggtattt tgtcaccatt catttttatc tggttgttct caagttcggt tacgagatcc
     2101 atttgtctat ctagttcaac ttggaaaatc aacgtatcag tcgggcggcc tcgcttatca
     2161 accaccaatt tcatattgct gtaagtgttt aaatctttac ttattggttt caaaacccat
     2221 tggttaagcc ttttaaactc atggtagtta ttttcaagca ttaacatgaa cttaaattca
     2281 tcaaggctaa tctctatatt tgccttgtga gttttctttt gtgttagttc ttttaataac
     2341 cactcataaa tcctcataga gtatttgttt tcaaaagact taacatgttc cagattatat
     2401 tttatgaatt tttttaactg gaaaagataa ggcaatatct cttcactaaa aactaattct
     2461 aatttttcgc ttgagaactt ggcatagttt gtccactgga aaatctcaaa gcctttaacc
     2521 aaaggattcc tgatttccac agttctcgtc atcagctctc tggttgcttt agctaataca
     2581 ccataagcat tttccctact gatgttcatc atctgagcgt attggttata agtgaacgat
     2641 accgtccgtt ctttccttgt agggttttca atcgtggggt tgagtagtgc cacacagcat
     2701 aaaattagct tggtttcatg ctccgttaag tcatagcgac taatcgctag ttcatttgct
     2761 ttgaaaacaa ctaattcaga catacatctc aattggtcta ggtgatttta atcactatac
     2821 caattgagat gggctagtca atgataatta ctagtccttt tcctttgagt tgtgggtatc
     2881 tgtaaattct gctagacctt tgctggaaaa cttgtaaatt ctgctagacc ctctgtaaat
     2941 tccgctagac ctttgtgtgt tttttttgtt tatattcaag tggttataat ttatagaata
     3001 aagaaagaat aaaaaaagat aaaaagaata gatcccagcc ctgtgtataa ctcactactt
     3061 tagtcagttc cgcagtatta caaaaggatg tcgcaaacgc tgtttgctcc tctacaaaac
     3121 agaccttaaa accctaaagg cttaagtagc gccgtcgcaa gctccggcaa atcgctgaat
     3181 attccttttg tctccgacca tcaggcacct gagtcgctgt ctttttcgtc acattcagtt
     3241 cgctgcgctc acggctctgg cagtgaatgg gggtaaatgg cactacaggc gccttttatg
     3301 gattcatgca aggaaactac ccataataca agaaaagccc gtcacgggct tctcagggcg
     3361 ttttatggcg ggtctgctat gtggtgctat ctgacttttt gctgttcagc agttcctgcc
     3421 ctctgatttt ccagtctgac cacttcggat tatcccgtga caggtcattc agactggcta
     3481 atgcacccag taaggcagcg gtatcatcaa caggcttacc cgtcttactg tccctagtgc
     3541 ttggattctc accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat
     3601 ggagttctga ggtcattact ggatctatca acaggagtcc aagcgagctc tcgaacccca
     3661 gagtcccgct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg
     3721 agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc
     3781 aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca
     3841 gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc
     3901 atgggtcacg acgagatcct cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc
     3961 ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc
     4021 catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc
     4081 cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg
     4141 agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct
     4201 tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca
     4261 cgatagccgc gctgcctcgt cctgcagttc attcagggca ccggacaggt cggtcttgac
     4321 aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat
     4381 tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc
     4441 gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatc
//
