LOCUS       sequence-492058-        6986 bp    DNA     circular SYN 06-NOV-2025
DEFINITION  Insert an N-terminal FRB-msGFP2 tag at VRG4 by popin-popout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6986)
  TITLE     Glick lab plasmids 2025
REFERENCE   2  (bases 1 to 6986)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: YIplac211-FRB-msGFP2-Vrg4
FEATURES             Location/Qualifiers
     source          1..6986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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)."
     primer_bind     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             732..1010
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             1029..1736
                     /codon_start=1
                     /product="photostable monomeric superfolder GFP (Valbuena 
                     et al., 2020)"
                     /label=msGFP2
                     /translation="MDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITGGSGGS"
     primer_bind     complement(3484..3501)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3484..3500)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3493..3515)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3709..3728)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3828..3850
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3968..4771)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(4772..4987)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4994..5012)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5080..5184
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5185..5253,5254..6045)
                     /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     complement(5403..5422)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6216..6804
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6705..6724
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6958..6975
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgt
      241 aatggaccat ggcaatgtgc gccatatata tatgcggctc ccttatgagt agaagaaaat
      301 atatgactgg tggtacaaag tggcatggtg gtatatagat gtaggaacac catttgagtt
      361 tgccataatg attaaatttc cctttttata aagaattagc cgcccataac acgaaaaata
      421 tcacagttga cgaaagaaga cacgtcgcct aaacgcgtaa tgaatcgcaa tatacgaaaa
      481 gctaaacggg tttttcagaa tcctcttacc cagccttgga taatagtttt ggccgaaaaa
      541 aaaaaaaaga aagaaaaaaa ttgaaatttc gcgatccgaa caaacaatga acagaaaaac
      601 agaatatata taacgctgcc aagcctattt cttgttgccg attaattgtt gacatcctct
      661 taattaccaa aagagcctaa gaaaacaaac acactaacca cacagtatct ttcgcccgaa
      721 tggcttctag aatcctctgg catgagatgt ggcatgaagg cctggaagag gcatctcgtt
      781 tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt gctggagccc ttgcatgcta
      841 tgatggaacg gggcccccag actctgaagg aaacatcctt taatcaggcc tatggtcgag
      901 atttaatgga ggcccaagag tggtgcagga agtacatgaa atcagggaat gtcaaggacc
      961 tcacccaagc ctgggacctc tattatcatg tgttccgacg aatctcaaag ggaggttctg
     1021 gaggttccat ggatagcact gagagcttgt tcacaggtgt tgttcctatc ttggtcgaat
     1081 tagacggtga cgtaaacggt cataagttca gtgttagagg tgaaggtgaa ggtgacgcta
     1141 ctaacggtaa attgacattg aagtttattt gtactacagg taaattgcca gttccttggc
     1201 caactttggt tactacattg acatacggtg ttcaatgttt ttcaagatac ccagatcata
     1261 tgaagcaaca tgatttcttt aagtctgcta tgccagaagg ttacgttcaa gaaagaacta
     1321 tcacttttaa agatgatggt acttacaaga caagagcaga ggttaagttc gaaggtgaca
     1381 ctttggttaa cagaatcgaa ttgaagggta tcgattttaa agaagatggt aatattttgg
     1441 gtcataaatt ggaatacaac tacaactcac ataacgttta cattacagct gataaacaaa
     1501 agaatggtat taaagcaaac ttcaagatca gacataatgt tgaagatggt tctgttcaat
     1561 tagcagatca ttaccaacaa aacactccaa ttggtgacgg tccagttttg ttaccagata
     1621 accattactt gtctacacaa tcaaaattgt ctaaagatcc aaacgaaaag agagatcata
     1681 tggttttgtt agaatttgtt actgctgctg gtatcacagg tggcagtggc ggttcttcag
     1741 aattgaaaac aggtcatgca ggccataacc cttgggcttc agttgccaat tccggtccga
     1801 tctctatttt atcctactgt ggttcctcta ttttaatgac ggtgactaac aagttcgtcg
     1861 tcaatttgaa ggatttcaac atgaactttg tcatgctttt cgtgcaatct ttggtttgta
     1921 ctataacctt gattatccta cgtatactgg gctatgcgaa gttccgttca ttaaacaaaa
     1981 cagacgccaa gaactggttc cctatttcct ttttactggt cttgatgatc tacacctctt
     2041 cgaaggcttt acaatacttg gctgttccaa tttacaccat tttcaagaat ttgactatta
     2101 tcttgattgc ttatggtgag gttctctttt ttggtggctc tgtcacctcc atggaattgt
     2161 catcattttt gttgatggtc ctttcttctg tcgttgcaac ttggggtgac cagcaagctg
     2221 tggctgccaa ggctgcttca ttggctgaag gagcagccgg tgctgttgcc tcctttaacc
     2281 caggttattt ctggatgttc accaactgta tcacttctgc attattcgtt cttataatga
     2341 gaaagagaat taagttaact aacttcaagg atttcgacac tatgttttac aacaatgttt
     2401 tggctctacc tattctattg ctgttttctt tctgtgtgga agattggtct tcagttaatt
     2461 tgaccaataa cttttctaac gattcgctaa ctgctatgat catcagtggt gttgcatccg
     2521 tcggtatttc ttactgttcc ggttggtgtg ttcgtgttac ttcgtctact acatattcga
     2581 tggtaggggc tttgaacaag ctgccaattg ccttgtctgg tttgattttc tttgatgctc
     2641 caagaaactt cttatctatt ctctccattt ttattggttt cctatcaggt attatttatg
     2701 ctgttgccaa acaaaagaag caacaagccc aacctttacg taaatgagaa cttacggggg
     2761 gtgcaattta tttttttttt ttggtttatt ttattttata gaggcatcta atgcaagtag
     2821 atttatatac aattatactt aaaattgata taccttagaa caggtcggac acaacgtgtg
     2881 gtaactccaa tatatcacta atgacaatag cgccttctgg tgtttgcccc aaaatttcat
     2941 ttacttcatt ctcaactaaa tgaatacatg ttttcatgcc caatttgata cctgtctcga
     3001 tgtttttccc actgtcatct ataaaataag cattctcgta tcttgccaac ccgctttcct
     3061 tcatggcttt ttcaaatgct ttaacatgtg gtttgcagac cagcgtatct gtcctagagt
     3121 agtcacaata tgtcaatcca tcaaataaat ccgctatacc caacaatctc aggcaacgta
     3181 ttgcgtggtt tttataagcg ttggtgaaaa gccacaattt atcaatcttt cccgattgtc
     3241 tcaatctcaa cagcatattc cttaatggta tatcaggttt caagatatct tgtagtggta
     3301 aggaatcatc cacaagccgg ttatattcca gtgcgttcac tttgtggaac attacaagtc
     3361 cccttatggc aagtccatac tctttgtagt aagagttatt caaaacgtga gcatcttcag
     3421 gtgatagttt caagtgtgtt tgaaagaacc tcagtataga ttgttgcata agatcgtgaa
     3481 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     3541 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     3601 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
     3661 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     3721 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     3781 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3841 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcg ggtaataact
     3901 gatataatta aattgaagct ctaatttgtg agtttagtat acatgcattt acttataata
     3961 cagtttttta gttttgctgg ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt
     4021 aacgttcacc ctctacctta gcatcccttc cctttgcaaa tagtcctctt ccaacaataa
     4081 taatgtcaga tcctgtagag accacatcat ccacggttct atactgttga cccaatgcgt
     4141 ctcccttgtc atctaaaccc acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc
     4201 ttccacccat gtctctttga gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa
     4261 tgtcaacagt acccttagta tattctccag tagataggga gcccttgcat gacaattctg
     4321 ctaacatcaa aaggcctcta ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc
     4381 taacaatacc tgggcccacc acaccgtgtg cattcgtaat gtctgcccat tctgctattc
     4441 tgtatacacc cgcagagtac tgcaatttga ctgtattacc aatgtcagca aattttctgt
     4501 cttcgaagag taaaaaattg tacttggcgg ataatgcctt tagcggctta actgtgccct
     4561 ccatggaaaa atcagtcaag atatccacat gtgtttttag taaacaaatt ttgggaccta
     4621 atgcttcaac taactccagt aattccttgg tggtacgaac atccaatgaa gcacacaagt
     4681 ttgtttgctt ttcgtgcatg atattaaata gcttggcagc aacaggacta ggatgagtag
     4741 cagcacgttc cttatatgta gctttcgaca tgatttatct tcgtttcctg catgtttttg
     4801 ttctgtgcag ttgggttaag aatactgggc aatttcatgt ttcttcaaca ctacatatgc
     4861 gtatatatac caatctaagt ctgtgctcct tccttcgttc ttccttctgt tcggagatta
     4921 ccgaatcaaa aaaatttcaa ggaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg
     4981 aattgaaaag ctggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     5041 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5101 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5161 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5221 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5281 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5341 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5401 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5461 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5521 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5581 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5641 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5701 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5761 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5821 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5881 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     5941 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6001 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6061 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6121 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6181 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6241 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6301 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6361 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6421 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6481 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6541 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6601 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6661 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6721 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     6781 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6841 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     6901 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     6961 gagtcagtga gcgaggaagc ggaaga
//
