LOCUS       sequence-203655-        4479 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 4479)
  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 4479)
  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 No Linker
FEATURES             Location/Qualifiers
     source          1..4479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     444..463
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     696..714
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     5'UTR           815..944
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             987..1700
                     /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(1017..1045)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1640..1660
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1741..1827
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2365..3315)
                     /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      3363..3585
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(4077..4171)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(4202..4479,1..517))
                     /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(4313..4332)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga
       61 caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga
      121 atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata
      181 ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata
      241 gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg
      301 tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca
      361 ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat
      421 cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg
      481 ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct catcctgtct
      541 cttgatcaga tcttgatccc ctgcgccatc agatccttgg cggcaagaaa gccatccagt
      601 ttactttgca gggcttccca accttaccag agggcgcccc agctggcaat tccgacgtct
      661 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
      721 gtcttcacct cgatgatatg aaaaataatt gttgcatcac ccgccaatgc gtggcttaat
      781 gcacatcaac ggtttgacgt acagaccatt aaagcagtgt agtaaggcaa gtcccttcaa
      841 gagttatcgt tgatacccct cgtagtgcac attcctttaa cgcttcaaaa tctgtaaagc
      901 acgccatatc gccgaaaggc acacttaatt attaaaggta atacactatg tccggtaaaa
      961 tgactggtat cgtaaaatgg ttcaacatga gtaaaggaga agaacttttc actggagttg
     1021 tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct gtcagtggag
     1081 agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc actactggaa
     1141 aactacctgt tccatggcca acacttgtca ctactttcgc gtatggtctt caatgctttg
     1201 cgagataccc agatcatatg aaacagcatg actttttcaa gagtgccatg cccgaaggtt
     1261 atgtacagga aagaactata tttttcaaag atgacgggaa ctacaagaca cgtgctgaag
     1321 tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt gattttaaag
     1381 aagatggaaa cattcttgga cacaaattgg aatacaacta taactcacac aatgtataca
     1441 tcatggcaga caaacaaaag aatggaatca aagttaactt caaaattaga cacaacattg
     1501 aagatggaag cgttcaacta gcagaccatt atcaacaaaa tactccaatt ggcgatggcc
     1561 ctgtcctttt accagacaac cattacctgt ccacacaatc tgccctttcg aaagatccca
     1621 acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg attacacatg
     1681 gcatggatga actatacaaa taaatgtcca gacctgcagg catgcaagct ctagaggcat
     1741 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     1801 gtgaacgctc tcctgagtag gacaaatccg ccgccctaga cctagggtac gggttttgct
     1861 gcccgcaaac gggctgttct ggtgttgcta gtttgttatc agaatcgcag atccggcttc
     1921 agccggtttg ccggctgaaa gcgctatttc ttccagaatt gccatgattt tttccccacg
     1981 ggaggcgtca ctggctcccg tgttgtcggc agctttgatt cgataagcag catcgcctgt
     2041 ttcaggctgt ctatgtgtga ctgttgagct gtaacaagtt gtctcaggtg ttcaatttca
     2101 tgttctagtt gctttgtttt actggtttca cctgttctat taggtgttac atgctgttca
     2161 tctgttacat tgtcgatctg ttcatggtga acagctttga atgcaccaaa aactcgtaaa
     2221 agctctgatg tatctatctt ttttacaccg ttttcatctg tgcatatgga cagttttccc
     2281 tttgatatgt aacggtgaac agttgttcta cttttgtttg ttagtcttga tgcttcactg
     2341 atagatacaa gagccataag aacctcagat ccttccgtat ttagccagta tgttctctag
     2401 tgtggttcgt tgtttttgcg tgagccatga gaacgaacca ttgagatcat acttactttg
     2461 catgtcactc aaaaattttg cctcaaaact ggtgagctga atttttgcag ttaaagcatc
     2521 gtgtagtgtt tttcttagtc cgttatgtag gtaggaatct gatgtaatgg ttgttggtat
     2581 tttgtcacca ttcattttta tctggttgtt ctcaagttcg gttacgagat ccatttgtct
     2641 atctagttca acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat caaccaccaa
     2701 tttcatattg ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc attggttaag
     2761 ccttttaaac tcatggtagt tattttcaag cattaacatg aacttaaatt catcaaggct
     2821 aatctctata tttgccttgt gagttttctt ttgtgttagt tcttttaata accactcata
     2881 aatcctcata gagtatttgt tttcaaaaga cttaacatgt tccagattat attttatgaa
     2941 tttttttaac tggaaaagat aaggcaatat ctcttcacta aaaactaatt ctaatttttc
     3001 gcttgagaac ttggcatagt ttgtccactg gaaaatctca aagcctttaa ccaaaggatt
     3061 cctgatttcc acagttctcg tcatcagctc tctggttgct ttagctaata caccataagc
     3121 attttcccta ctgatgttca tcatctgagc gtattggtta taagtgaacg ataccgtccg
     3181 ttctttcctt gtagggtttt caatcgtggg gttgagtagt gccacacagc ataaaattag
     3241 cttggtttca tgctccgtta agtcatagcg actaatcgct agttcatttg ctttgaaaac
     3301 aactaattca gacatacatc tcaattggtc taggtgattt taatcactat accaattgag
     3361 atgggctagt caatgataat tactagtcct tttcctttga gttgtgggta tctgtaaatt
     3421 ctgctagacc tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa attccgctag
     3481 acctttgtgt gttttttttg tttatattca agtggttata atttatagaa taaagaaaga
     3541 ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat aactcactac tttagtcagt
     3601 tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa acagacctta
     3661 aaaccctaaa ggcttaagta gcaccctcgc aagctcgggc aaatcgctga atattccttt
     3721 tgtctccgac catcaggcac ctgagtcgct gtctttttcg tgacattcag ttcgctgcgc
     3781 tcacggctct ggcagtgaat gggggtaaat ggcactacag gcgcctttta tggattcatg
     3841 caaggaaact acccataata caagaaaagc ccgtcacggg cttctcaggg cgttttatgg
     3901 cgggtctgct atgtggtgct atctgacttt ttgctgttca gcagttcctg ccctctgatt
     3961 ttccagtctg accacttcgg attatcccgt gacaggtcat tcagactggc taatgcaccc
     4021 agtaaggcag cggtatcatc aacaggctta cccgtcttac tgtccctagt gcttggattc
     4081 tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct
     4141 gaggtcatta ctggatctat caacaggagt ccaagcgagc tctcgaaccc cagagtcccg
     4201 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
     4261 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
     4321 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
     4381 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca
     4441 cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcc
//
