LOCUS       sequence-205131-        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     complement(79..98)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     689..708
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     941..959
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     5'UTR           1060..1189
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             1247..1960
                     /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(1277..1305)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1900..1920
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2001..2087
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2625..3575)
                     /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      3623..3845
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(4337..4431)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(4462..4494,1..762))
                     /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"
ORIGIN
        1 gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc
       61 gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc
      121 cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt
      181 cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt
      241 gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg
      301 atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg
      361 gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat
      421 ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc
      481 caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac
      541 gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc
      601 ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc
      661 ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca
      721 agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc
      781 tgtctcttga tcagatcttg atcccctgcg ccatcagatc cttggcggca agaaagccat
      841 ccagtttact ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccga
      901 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
      961 ctttcgtctt cacctcgatg atatgaaaaa taattgttgc atcacccgcc aatgcgtggc
     1021 ttaatgcaca tcaacggttt gacgtacaga ccattaaagc agtgtagtaa ggcaagtccc
     1081 ttcaagagtt atcgttgata cccctcgtag tgcacattcc tttaacgctt caaaatctgt
     1141 aaagcacgcc atatcgccga aaggcacact taattattaa aggtaataca ctatgtccgg
     1201 taaaatgact ggtatcgtaa aatggttcaa cggtggaggt ggaagcatga gtaaaggaga
     1261 agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca
     1321 caaattttct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa
     1381 atttatttgc actactggaa aactacctgt tccatggcca acacttgtca ctactttcgc
     1441 gtatggtctt caatgctttg cgagataccc agatcatatg aaacagcatg actttttcaa
     1501 gagtgccatg cccgaaggtt atgtacagga aagaactata tttttcaaag atgacgggaa
     1561 ctacaagaca cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt
     1621 aaaaggtatt gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacta
     1681 taactcacac aatgtataca tcatggcaga caaacaaaag aatggaatca aagttaactt
     1741 caaaattaga cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa
     1801 tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc
     1861 tgccctttcg aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac
     1921 agctgctggg attacacatg gcatggatga actatacaaa taaatgtcca gacctgcagg
     1981 catgcaagct ctagaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     2041 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgccctaga
     2101 cctagggtac gggttttgct gcccgcaaac gggctgttct ggtgttgcta gtttgttatc
     2161 agaatcgcag atccggcttc agccggtttg ccggctgaaa gcgctatttc ttccagaatt
     2221 gccatgattt tttccccacg ggaggcgtca ctggctcccg tgttgtcggc agctttgatt
     2281 cgataagcag catcgcctgt ttcaggctgt ctatgtgtga ctgttgagct gtaacaagtt
     2341 gtctcaggtg ttcaatttca tgttctagtt gctttgtttt actggtttca cctgttctat
     2401 taggtgttac atgctgttca tctgttacat tgtcgatctg ttcatggtga acagctttga
     2461 atgcaccaaa aactcgtaaa agctctgatg tatctatctt ttttacaccg ttttcatctg
     2521 tgcatatgga cagttttccc tttgatatgt aacggtgaac agttgttcta cttttgtttg
     2581 ttagtcttga tgcttcactg atagatacaa gagccataag aacctcagat ccttccgtat
     2641 ttagccagta tgttctctag tgtggttcgt tgtttttgcg tgagccatga gaacgaacca
     2701 ttgagatcat acttactttg catgtcactc aaaaattttg cctcaaaact ggtgagctga
     2761 atttttgcag ttaaagcatc gtgtagtgtt tttcttagtc cgttatgtag gtaggaatct
     2821 gatgtaatgg ttgttggtat tttgtcacca ttcattttta tctggttgtt ctcaagttcg
     2881 gttacgagat ccatttgtct atctagttca acttggaaaa tcaacgtatc agtcgggcgg
     2941 cctcgcttat caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt acttattggt
     3001 ttcaaaaccc attggttaag ccttttaaac tcatggtagt tattttcaag cattaacatg
     3061 aacttaaatt catcaaggct aatctctata tttgccttgt gagttttctt ttgtgttagt
     3121 tcttttaata accactcata aatcctcata gagtatttgt tttcaaaaga cttaacatgt
     3181 tccagattat attttatgaa tttttttaac tggaaaagat aaggcaatat ctcttcacta
     3241 aaaactaatt ctaatttttc gcttgagaac ttggcatagt ttgtccactg gaaaatctca
     3301 aagcctttaa ccaaaggatt cctgatttcc acagttctcg tcatcagctc tctggttgct
     3361 ttagctaata caccataagc attttcccta ctgatgttca tcatctgagc gtattggtta
     3421 taagtgaacg ataccgtccg ttctttcctt gtagggtttt caatcgtggg gttgagtagt
     3481 gccacacagc ataaaattag cttggtttca tgctccgtta agtcatagcg actaatcgct
     3541 agttcatttg ctttgaaaac aactaattca gacatacatc tcaattggtc taggtgattt
     3601 taatcactat accaattgag atgggctagt caatgataat tactagtcct tttcctttga
     3661 gttgtgggta tctgtaaatt ctgctagacc tttgctggaa aacttgtaaa ttctgctaga
     3721 ccctctgtaa attccgctag acctttgtgt gttttttttg tttatattca agtggttata
     3781 atttatagaa taaagaaaga ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat
     3841 aactcactac tttagtcagt tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct
     3901 cctctacaaa acagacctta aaaccctaaa ggcttaagta gcaccctcgc aagctcgggc
     3961 aaatcgctga atattccttt tgtctccgac catcaggcac ctgagtcgct gtctttttcg
     4021 tgacattcag ttcgctgcgc tcacggctct ggcagtgaat gggggtaaat ggcactacag
     4081 gcgcctttta tggattcatg caaggaaact acccataata caagaaaagc ccgtcacggg
     4141 cttctcaggg cgttttatgg cgggtctgct atgtggtgct atctgacttt ttgctgttca
     4201 gcagttcctg ccctctgatt ttccagtctg accacttcgg attatcccgt gacaggtcat
     4261 tcagactggc taatgcaccc agtaaggcag cggtatcatc aacaggctta cccgtcttac
     4321 tgtccctagt gcttggattc tcaccaataa aaaacgcccg gcggcaaccg agcgttctga
     4381 acaaatccag atggagttct gaggtcatta ctggatctat caacaggagt ccaagcgagc
     4441 tctcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga aggc
//
