LOCUS       sequence-433682-        6183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Introduction of a bicistronic expression cassette of yEGFP and 
            E2Crimson under the control of the SkGAL2 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6183)
  AUTHORS   Lu Z, Shen Q, Bandari NC, Evans S, McDonnell L, Liu L, Jin W, 
            Luna-Flores CH, Collier T, Talbo G, McCubbin T, Esquirol L, Myers C,
            Trau M, Dumsday G, Speight R, Howard CB, Vickers CE, Peng B
  TITLE     LowTempGAL: a highly responsive low temperature-inducible GAL system
            in Saccharomyces cerevisiae.
  JOURNAL   Nucleic Acids Res. 2024 May 29:gkae460. doi: 10.1093/nar/gkae460.
  PUBMED    38808673
REFERENCE   2  (bases 1 to 6183)
  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"; journalName: "Nucleic 
            Acids Res. 2024 May 29:gkae460. doi: 10.1093/nar/gkae460."
COMMENT     Sequence Label: pILGFP4QE2Crimson
FEATURES             Location/Qualifiers
     source          1..6183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(244..260)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(244..260)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(257..279)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    268..284
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(292..298,299..316,317..322))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    337..358
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(475..492)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(646..1234)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(726..745)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1405..2196,2197..2265))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2028..2047
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2266..2370)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2438..2456
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2494..2516)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2616..2635
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2829..2851
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2843..2860
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2844..2860
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3974..4007)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             4734..5444
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
ORIGIN
        1 gatcttaaac tagtaggact atttgcaaag ggaagggatg ctaaggtaga gggtgaacgt
       61 tacagaaaag caggctggga agcatatttg agaagatgcg gccagcaaaa ctaaaaaact
      121 gtattataag taaatgcatg tatactaaac tcacaaatta gagcttcaat ttaattatat
      181 cagttattac ccgggaatct cggtcgtaat gatttaaatg catgcaagct tggcgtaatc
      241 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
      301 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
      361 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
      421 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
      481 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
      541 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
      601 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
      661 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
      721 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
      781 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
      841 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
      901 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
      961 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     1021 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     1081 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     1141 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     1201 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     1261 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     1321 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     1381 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     1441 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     1501 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     1561 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     1621 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     1681 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     1741 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     1801 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     1861 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     1921 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     1981 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     2041 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     2101 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     2161 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     2221 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     2281 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     2341 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     2401 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     2461 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     2521 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     2581 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     2641 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     2701 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     2761 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     2821 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattcattt aaattttttt
     2881 gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg agcacagact tagattggta
     2941 tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc cagtattctt aacccaactg
     3001 cacagaacaa aaacctgcag gaaacgaaga taaaacatgt ccacaaaatc atataccagt
     3061 agagctgaga ctcatgcaag tccggttgca tcgaaacttt tacgtttaat ggatgaaaag
     3121 aagaccaatt tgtgtgcttc tcttgacgtt cgttcgactg atgagctatt gaaacttgtt
     3181 gaaacgttgg gtccatacat ttgccttttg aaaacacacg ttgatatctt ggatgatttc
     3241 agttatgagg gtactgtcgt tccattgaaa gcattggcag agaaatacaa gttcttgata
     3301 tttgaggaca gaaaattcgc cgatatcggt aacacagtca aattacaata tacatcgggc
     3361 gtttaccgta tcgcagaatg gtctgatatc accaacgccc acggggttac tggtgctggt
     3421 attgttgctg gcttgaaaca aggtgcgcaa gaggtcacca aagaaccaag gggattattg
     3481 atgcttgctg aattgtcttc caagggttct ctagcacacg gtgaatatac taagggtacc
     3541 gttgatattg caaagagtga taaagatttc gttattgggt tcattgctca gaacgatatg
     3601 ggaggaagag aagaagggtt tgattggcta atcatgaccc caggtgtagg tttagacgac
     3661 aaaggcgatg cattgggtca gcagtacaga accgtcgacg aagttgtaag tggtggatca
     3721 gatatcatca ttgttggcag aggacttttc gccaagggta gagatcctaa ggttgaaggt
     3781 gaaagataca gaaatgctgg atgggaagcg taccaaaaga gaatcagcgc tccccattaa
     3841 ttatacagga aacttaatag aacaaatcac atatttaatc taatagccac ctgcattggc
     3901 acggtgcaac actacttcaa cttcatctta caaaaagatc acgtgatctg ttgtattggg
     3961 atctctagac ctaataactt cgtatagcat acattatacg aagttatatt aagggttgct
     4021 cgagtaaacc aattttattt gaacttgccc cgctaatcgt acagacgtac cagatccagg
     4081 tcataacggt gccattagaa ggccgtagaa tagacccact aaagctatca caggcaacct
     4141 tgcatccaag tctaattaaa cggagcagaa tctgttccag taatgtataa agaattaact
     4201 accggaaaag cgtcttccgg ggcggctcaa ccggattaca ttcttttttc ttttttgcgg
     4261 ccagcattcg agtcacgtgc tcaaatactg aggaaggggt tcttccggta ggcaccggcg
     4321 ctcactcttc cgtgcggcac actccccgca gttcaggggc catgtgcctg tgagagtatt
     4381 tccgcaggcc atagttccgg ggtggaccac tccgagctaa tgctaaagta agcccttccc
     4441 aacttgaaat aggaacaaaa gtcaattatg ctcctcctgt aaagctaaac gtgtaaagat
     4501 tttagagtga gacgttgtta gcagtcgttg ttcaagggag ggatgagttg aggaataaaa
     4561 tgcggaaaaa gtgtgggtta gacagacata tataaactct agccattcgg ctgaaatata
     4621 tctgagttca tttgtgataa cttttttgat tacttgttca tatatccaca agaacaacga
     4681 ctattaaaca atagaaaata gtataataaa aaaagttttt tacaatggga tcctctaaag
     4741 gtgaagaatt attcactggt gttgtcccaa ttttggttga attagatggt gatgttaatg
     4801 gtcacaaatt ttctgtctcc ggtgaaggtg aaggtgatgc tacttacggt aaattgacct
     4861 taaaatttat ttgtactact ggtaaattgc cagttccatg gccaacctta gtcactactt
     4921 tcggttatgg tgttcaatgt tttgcgagat acccagatca tatgaaacaa catgactttt
     4981 tcaagtctgc catgccagaa ggttatgttc aagaaagaac tatttttttc aaagatgacg
     5041 gtaactacaa gaccagagct gaagtcaagt ttgaaggtga taccttagtt aatagaatcg
     5101 aattaaaagg tattgatttt aaagaagatg gtaacatttt aggtcacaaa ttggaataca
     5161 actataactc tcacaatgtt tacatcatgg ctgacaaaca aaagaatggt atcaaagtta
     5221 acttcaaaat tagacacaac attgaagatg gttctgttca attagctgac cattatcaac
     5281 aaaatactcc aattggtgat ggtccagtct tgttaccaga caaccattac ttatccactc
     5341 aatctgcctt atccaaagat ccaaacgaaa agagagacca catggtcttg ttagaatttg
     5401 ttactgctgc tggtattacc catggtatgg atgaattgta caaaagtgct actaacttct
     5461 ctttgttgaa gttggctggt gacgttgaac ttaacccagg tccagactct accgaaaacg
     5521 ttatcaaacc attcatgaga ttcaaggtcc acatggaagg ttctgttaac ggtcatgaat
     5581 ttgaaatcga aggtgtcggt gaaggtaaac catacgaagg tactcaaact gctaagttgc
     5641 aagttacaaa aggtggtcca ttgccatttg cttgggatat tttgtctcca caattcttct
     5701 atggttccaa ggcttacatc aaacatccag ctgatattcc agactacttg aagcaatcat
     5761 ttccagaggg ttttaagtgg gaaagagtca tgaacttcga agatggtggt gttgttactg
     5821 ttacccaaga ttcttcattg caagacggta ctttgatcta ccacgttaag ttcattggtg
     5881 tcaactttcc atctgatggt ccagtcatgc aaaagaaaac tttaggttgg gaaccatcca
     5941 ccgaaagaaa ttatccaaga gatggtgttt tgaagggtga aaatcacatg gccttgaaat
     6001 tgaaaggtgg tggtcattat ttgtgcgagt tcaagtctat ctacatggct aagaaaccag
     6061 ttaagttgcc aggttatcac tacgttgatt acaagttgga tattacctct cacaacgaag
     6121 attacaccgt tgtcgaacaa tacgaaagag ctgaagctag acatcacttg ttccaatgat
     6181 aaa
//
