LOCUS       sequence-163663-        6121 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-FKBP(M)x4 in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6121)
  AUTHORS   Barrero JJ, Papanikou E, Casler JC, Day KJ, Glick BS
  TITLE     An improved reversibly dimerizing mutant of the FK506-binding 
            protein FKBP.
  JOURNAL   Cell Logist. 2016 Jun 24;6(3):e1204848. eCollection 2016 Jul-Sep.
  PUBMED    27738551
REFERENCE   2  (bases 1 to 6121)
  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: "Cell 
            Logist."; date: "2016-06-24"; volume: "6(3)"; pages: "e1204848. 
            eCollection 2016 Jul-Sep"
COMMENT     Sequence Label: EGFP-FKBP(M)x4
FEATURES             Location/Qualifiers
     source          1..6121
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..29)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(183..771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(263..282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(942..1733,1734..1802))
                     /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     1565..1584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1803..1907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1975..1993
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1995..2096
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             2097..2771
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     primer_bind     complement(2890..2912)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3012..3031
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3155..3585
                     /gene="S. cerevisiae TPI1"
                     /label=TPI1 promoter
                     /note="strong constitutive promoter for yeast triose 
                     phosphate isomerase"
     CDS             join(3596..3598,3599..3601,3602..4312)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3596..3598,3599..3601,3602..4312)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3641..3662)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3902..3921)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4249..4270
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4325..4645
                     /codon_start=1
                     /product="dimeric F36M mutant of FK506-binding protein 
                     FKBP12"
                     /label=FKBP-M
                     /note="dimerization can be reversed by ligand binding"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKMDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             4652..4972
                     /codon_start=1
                     /product="dimeric F36M mutant of FK506-binding protein 
                     FKBP12"
                     /label=FKBP-M
                     /note="dimerization can be reversed by ligand binding"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKMDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             4979..5299
                     /codon_start=1
                     /product="dimeric F36M mutant of FK506-binding protein 
                     FKBP12"
                     /label=FKBP-M
                     /note="dimerization can be reversed by ligand binding"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKMDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             5306..5629
                     /codon_start=1
                     /product="dimeric F36M mutant of FK506-binding protein 
                     FKBP12"
                     /label=FKBP-M
                     /note="dimerization can be reversed by ligand binding"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKMDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     terminator      5636..5883
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5652..5670)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5902..5918)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5902..5918)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5915..5937)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5926..5942
                     /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(5950..5956,5957..5974,5975..5980))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5995..6016
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
       61 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      601 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
      661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1081 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1801 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1861 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1921 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1981 cgtatcacga ggccaattcg gtcgaaaaaa gaaaaggaga gggccaagag ggagggcatt
     2041 ggtgactatt gagcacgtga gtatacgtga ttaagcacac aaaggcagct tggagtatgt
     2101 ctgttattaa tttcacaggt agttctggtc cattggtgaa agtttgcggc ttgcagagca
     2161 cagaggccgc agaatgtgca ctagattccg atgctgactt gctgggtatt atatgtgtgc
     2221 ccaatagaaa gagaacaatt gacccggtta ttgcaaggaa aatttcaagt cttgtaaaag
     2281 catataaaaa tagttcaggc actccgaaat acttggttgg cgtgtttcgt aatcaaccta
     2341 aggaggatgt tttggctctg gtcaatgatt acggcattga tatcgtccaa ctgcatggag
     2401 atgagtcgtg gcaagaatac caagagttcc tcggtttgcc agttattaaa agactcgtat
     2461 ttccaaaaga ctgcaacata ctactcagtg cagcttcaca gaaacctcat tcgtttattc
     2521 ccttgtttga ttcagaagca ggtgggacag gtgaactttt ggattggaac tcgatttctg
     2581 actgggttgg aaggcaagag agccccgaga gcttacattt tatgttagct ggtggactga
     2641 cgccagaaaa tgttggtgat gcgcttagat taaatggcgt tattggtgtt gatgtaagcg
     2701 gaggtgtgga gacaaatggt gtaaaagact ctaacaaaat agcaaatttc gtcaaaaatg
     2761 ctaagaaata ggttattact gagtagtatt tatttaagta ttgtttgtgc acttgcctgc
     2821 aagccttttg aaaagcaagc ataaaagatc gccctttcgt ctcgcgcgtt tcggtgatga
     2881 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     2941 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
     3001 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     3061 accgcacaga tgcgtaagga gaaaataccg catcaggcgg accttaatac attcagacac
     3121 ttctgcggta tcaccctact tattcccttc gagattatat ctaggaaccc atcaggttgg
     3181 tggaagatta cccgttctaa gacttttcag cttcctctat tgatgttaca cctggacacc
     3241 ccttttctgg catccagttt ttaatcttca gtggcatgtg agattctccg aaattaatta
     3301 aagcaatcac acaattctct cggataccac ctcggttgaa actgacaggt ggtttgttac
     3361 gcatgctaat gcaaaggagc ctatatacct ttggctcggc tgctgtaaca gggaatataa
     3421 agggcagcat aatttaggag tttagtgaac ttgcaacatt tactattttc ccttcttacg
     3481 taaatatttt tctttttaat tctaaatcaa tctttttcaa ttttttgttt gtattctttt
     3541 cttgcttaaa tctataacta caaaaaacac atacataaac taaaagaatt caaaaatggt
     3601 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     3661 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     3721 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     3781 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     3841 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     3901 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     3961 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     4021 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     4081 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     4141 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     4201 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     4261 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agggatcctc
     4321 tagaggagtg caggtggaaa ccatctcccc aggagacggg cgcaccttcc ccaagcgcgg
     4381 ccagacctgc gtggtgcact acaccgggat gcttgaagat ggaaagaaaa tggattcctc
     4441 ccgggacaga aacaagccct ttaagtttat gctaggcaag caggaggtga tccgaggctg
     4501 ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc aaactgacta tatctccaga
     4561 ttatgcctat ggtgccactg ggcacccagg catcatccca ccacatgcca ctctcgtctt
     4621 cgatgtggag cttctaaaac tggaaactag aggagtgcag gtggaaacca tctccccagg
     4681 agacgggcgc accttcccca agcgcggcca gacctgcgtg gtgcactaca ccgggatgct
     4741 tgaagatgga aagaaaatgg attcctcccg ggacagaaac aagcccttta agtttatgct
     4801 aggcaagcag gaggtgatcc gaggctggga agaaggggtt gcccagatga gtgtgggtca
     4861 gagagccaaa ctgactatat ctccagatta tgcctatggt gccactgggc acccaggcat
     4921 catcccacca catgccactc tcgtcttcga tgtggagctt ctaaaactgg aaactagagg
     4981 agtgcaggtg gaaaccatct ccccaggaga cgggcgcacc ttccccaagc gcggccagac
     5041 ctgcgtggtg cactacaccg ggatgcttga agatggaaag aaaatggatt cctcccggga
     5101 cagaaacaag ccctttaagt ttatgctagg caagcaggag gtgatccgag gctgggaaga
     5161 aggggttgcc cagatgagtg tgggtcagag agccaaactg actatatctc cagattatgc
     5221 ctatggtgcc actgggcacc caggcatcat cccaccacat gccactctcg tcttcgatgt
     5281 ggagcttcta aaactggaaa ctagaggagt gcaggtggaa accatctccc caggagacgg
     5341 gcgcaccttc cccaagcgcg gccagacctg cgtggtgcac tacaccggga tgcttgaaga
     5401 tggaaagaaa atggattcct cccgggacag aaacaagccc tttaagttta tgctaggcaa
     5461 gcaggaggtg atccgaggct gggaagaagg ggttgcccag atgagtgtgg gtcagagagc
     5521 caaactgact atatctccag attatgccta tggtgccact gggcacccag gcatcatccc
     5581 accacatgcc actctcgtct tcgatgtgga gcttctaaaa ctggaataaa ctagatcatg
     5641 taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag
     5701 gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta
     5761 ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca
     5821 tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt
     5881 tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     5941 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     6001 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6061 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6121 c
//
