LOCUS       sequence-199788-        4500 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 4500)
  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 4500)
  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..4500
                     /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             440..1153
                     /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(470..498)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1093..1113
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1194..1280
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1818..2768)
                     /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      2816..3038
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3530..3624)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(3766..3785)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4397..4416
                     /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 tggttcaaca tgagtaaagg agaagaactt ttcactggag ttgtcccaat
      481 tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg gagagggtga
      541 aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg gaaaactacc
      601 tgttccatgg ccaacacttg tcactacttt cgcgtatggt cttcaatgct ttgcgagata
      661 cccagatcat atgaaacagc atgacttttt caagagtgcc atgcccgaag gttatgtaca
      721 ggaaagaact atatttttca aagatgacgg gaactacaag acacgtgctg aagtcaagtt
      781 tgaaggtgat acccttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg
      841 aaacattctt ggacacaaat tggaatacaa ctataactca cacaatgtat acatcatggc
      901 agacaaacaa aagaatggaa tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg
      961 aagcgttcaa ctagcagacc attatcaaca aaatactcca attggcgatg gccctgtcct
     1021 tttaccagac aaccattacc tgtccacaca atctgccctt tcgaaagatc ccaacgaaaa
     1081 gagagaccac atggtccttc ttgagtttgt aacagctgct gggattaccc atggtatgga
     1141 tgaattgtac aaataaatgt ccagacctgc aggcatgcaa gctctagagg catcaaataa
     1201 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     1261 ctctcctgag taggacaaat ccgccgccct agacctaggg tacgggtttt gctgcccgca
     1321 aacgggctgt tctggtgttg ctagtttgtt atcagaatcg cagatccggc ttcagccggt
     1381 ttgccggctg aaagcgctat ttcttccaga attgccatga ttttttcccc acgggaggcg
     1441 tcactggctc ccgtgttgtc ggcagctttg attcgataag cagcatcgcc tgtttcaggc
     1501 tgtctatgtg tgactgttga gctgtaacaa gttgtctcag gtgttcaatt tcatgttcta
     1561 gttgctttgt tttactggtt tcacctgttc tattaggtgt tacatgctgt tcatctgtta
     1621 cattgtcgat ctgttcatgg tgaacagctt tgaatgcacc aaaaactcgt aaaagctctg
     1681 atgtatctat cttttttaca ccgttttcat ctgtgcatat ggacagtttt ccctttgata
     1741 tgtaacggtg aacagttgtt ctacttttgt ttgttagtct tgatgcttca ctgatagata
     1801 caagagccat aagaacctca gatccttccg tatttagcca gtatgttctc tagtgtggtt
     1861 cgttgttttt gcgtgagcca tgagaacgaa ccattgagat catacttact ttgcatgtca
     1921 ctcaaaaatt ttgcctcaaa actggtgagc tgaatttttg cagttaaagc atcgtgtagt
     1981 gtttttctta gtccgttatg taggtaggaa tctgatgtaa tggttgttgg tattttgtca
     2041 ccattcattt ttatctggtt gttctcaagt tcggttacga gatccatttg tctatctagt
     2101 tcaacttgga aaatcaacgt atcagtcggg cggcctcgct tatcaaccac caatttcata
     2161 ttgctgtaag tgtttaaatc tttacttatt ggtttcaaaa cccattggtt aagcctttta
     2221 aactcatggt agttattttc aagcattaac atgaacttaa attcatcaag gctaatctct
     2281 atatttgcct tgtgagtttt cttttgtgtt agttctttta ataaccactc ataaatcctc
     2341 atagagtatt tgttttcaaa agacttaaca tgttccagat tatattttat gaattttttt
     2401 aactggaaaa gataaggcaa tatctcttca ctaaaaacta attctaattt ttcgcttgag
     2461 aacttggcat agtttgtcca ctggaaaatc tcaaagcctt taaccaaagg attcctgatt
     2521 tccacagttc tcgtcatcag ctctctggtt gctttagcta atacaccata agcattttcc
     2581 ctactgatgt tcatcatctg agcgtattgg ttataagtga acgataccgt ccgttctttc
     2641 cttgtagggt tttcaatcgt ggggttgagt agtgccacac agcataaaat tagcttggtt
     2701 tcatgctccg ttaagtcata gcgactaatc gctagttcat ttgctttgaa aacaactaat
     2761 tcagacatac atctcaattg gtctaggtga ttttaatcac tataccaatt gagatgggct
     2821 agtcaatgat aattactagt ccttttcctt tgagttgtgg gtatctgtaa attctgctag
     2881 acctttgctg gaaaacttgt aaattctgct agaccctctg taaattccgc tagacctttg
     2941 tgtgtttttt ttgtttatat tcaagtggtt ataatttata gaataaagaa agaataaaaa
     3001 aagataaaaa gaatagatcc cagccctgtg tataactcac tactttagtc agttccgcag
     3061 tattacaaaa ggatgtcgca aacgctgttt gctcctctac aaaacagacc ttaaaaccct
     3121 aaaggcttaa gtagcgccgt cgcaagctcc ggcaaatcgc tgaatattcc ttttgtctcc
     3181 gaccatcagg cacctgagtc gctgtctttt tcgtcacatt cagttcgctg cgctcacggc
     3241 tctggcagtg aatgggggta aatggcacta caggcgcctt ttatggattc atgcaaggaa
     3301 actacccata atacaagaaa agcccgtcac gggcttctca gggcgtttta tggcgggtct
     3361 gctatgtggt gctatctgac tttttgctgt tcagcagttc ctgccctctg attttccagt
     3421 ctgaccactt cggattatcc cgtgacaggt cattcagact ggctaatgca cccagtaagg
     3481 cagcggtatc atcaacaggc ttacccgtct tactgtccct agtgcttgga ttctcaccaa
     3541 taaaaaacgc ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt tctgaggtca
     3601 ttactggatc tatcaacagg agtccaagcg agctctcgaa ccccagagtc ccgctcagaa
     3661 gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta
     3721 aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc
     3781 caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga
     3841 aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag
     3901 atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc
     3961 ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc
     4021 tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg
     4081 cagccgccgg cggcaagaaa gccatccagg cattgcatca gccatgatgg atactttctc
     4141 ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca
     4201 gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc
     4261 cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt
     4321 cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca
     4381 gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga
     4441 acctgcgtgc aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc
//
