LOCUS       sequence-232760-        6256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast integrative vector for the expression of 2xGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6256)
  AUTHORS   Delarue M, Brittingham GP, Pfeffer S, Surovtsev IV, Pinglay S, 
            Kennedy KJ, Schaffer M, Gutierrez JI, Sang D, Poterewicz G, Chung 
            JK, Plitzko JM, Groves JT, Jacobs-Wagner C, Engel BD, Holt LJ
  TITLE     mTORC1 Controls Phase Separation and the Biophysical Properties of 
            the Cytoplasm by Tuning Crowding.
  JOURNAL   Cell. 2018 Jul 12;174(2):338-349.e20. doi: 
            10.1016/j.cell.2018.05.042. Epub 2018 Jun 21.
  PUBMED    29937223
REFERENCE   2  (bases 1 to 6256)
  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.cell.2018.05.042"; journalName: "Cell"; date: 
            "2018-07-12- 12"; volume: "174"; issue: "2"; pages: "338-349.e20"
COMMENT     Sequence Label: pRS306-pHIS3-2xGFP-tURA3
FEATURES             Location/Qualifiers
     source          1..6256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /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"
     rep_origin      complement(1344..1799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1481..1502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1693..1712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1925..1947
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1939..1956
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1940..1956
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1963..1982
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1963..1981
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2031..2217
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             2265..2975
                     /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="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     2915..2935
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2997..3710
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGIDELYK"
     promoter        complement(3996..4014)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4035..4051)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4035..4051)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4048..4070)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4059..4075
                     /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(4083..4089,4090..4107,4108..4113))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4128..4149
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4266..4283)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4437..5025)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4517..4536)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5196..5987,5988..6056))
                     /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     5819..5838
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6057..6161)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6229..6247
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt
     1081 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctatgcggtg tgaaataccg cacagatgcg
     1321 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     1381 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1441 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1501 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1561 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1621 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1681 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     1741 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     1801 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     1861 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     1921 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag
     1981 ggcgaattgg agctccaccg cggtggcggc cgcattactc ttggcctcct ctagtacact
     2041 ctatattttt ttatgcctcg gtaatgattt tcgttttttt tttccaccta gcggatgact
     2101 cttttttttt cttagcgatt ggcattatca cataatgaat tatacattat ataaagtaat
     2161 gtgatttcta cgaagaatat actaaaaaat gagcaggcaa gataaacgaa ggcaaagagc
     2221 tcggtacccg gggatccact agtatggcca gtgaattcga gctcagtaaa ggagaagaac
     2281 ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat
     2341 tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc cttaaattta
     2401 tttgcactac tggaaaacta cctgttccat ggccaacact tgtcactact ttcacttatg
     2461 gtgttcaatg cttttcaaga tacccagatc atatgaaacg gcatgacttt ttcaagagtg
     2521 ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac gggaactaca
     2581 agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc gagttaaaag
     2641 gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac aactataact
     2701 cacacaatgt atacatcatg gcagacaaac aaaagaatgg aatcaaagtt aacttcaaaa
     2761 ttagacacaa cattgaagat ggaagcgttc aactagcaga ccattatcaa caaaatactc
     2821 caattggcga tggccctgtc cttttaccag acaaccatta cctgtccaca caatctgccc
     2881 tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg
     2941 ctgggattac acatggcatg gatgaactat acaaaggagc gccgggagca ggtgcatcta
     3001 aaggtgaaga attattcact ggtgttgtcc caattttggt tgaattagat ggtgatgtta
     3061 atggtcacaa attttctgtc tccggtgaag gtgaaggtga tgctacttac ggtaaattga
     3121 ccttaaaatt tatttgtact actggtaaat tgccagttcc atggccaacc ttagtcacta
     3181 ctttaactta tggtgttcaa tgtttttcta gatacccaga tcatatgaaa caacatgact
     3241 ttttcaagtc tgccatgcca gaaggttatg ttcaagaaag aactattttt ttcaaagatg
     3301 acggtaacta caagaccaga gctgaagtca agtttgaagg tgatacctta gttaatagaa
     3361 tcgaattaaa aggtattgat tttaaagaag atggtaacat tttaggtcac aaattggaat
     3421 acaactataa ctctcacaat gtttacatca tggctgacaa acaaaagaat ggtatcaaag
     3481 ttaacttcaa aattagacac aacattgaag atggttctgt tcaattagct gaccattatc
     3541 aacaaaatac tccaattggt gatggtccag tcttgttacc agacaaccat tacttatcca
     3601 ctcaatctgc cttatccaaa gatccaaacg aaaagagaga ccacatggtc ttgttagaat
     3661 ttgttactgc tgctggtatt acccatggta ttgatgaatt gtacaaataa ggcgcgccat
     3721 tataagtaaa tgcatgtata ctaaactcac aaattagagc ttcaatttaa ttatatcagt
     3781 tattacccgg gaatctcggt cgtaatgatt tctataatga cgaaaaaaaa aaaattggaa
     3841 agaaaaagct tcatggcctt tataaaaagg aactatccaa tacctcgcca gaaccaagta
     3901 acagtatttt acggggcaca aatcaagaac aataagacag gactgtaaag atggacgcat
     3961 agcctcgagg gggggcccgg taccagcttt tgttcccttt agtgagggtt aatttcgagc
     4021 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4081 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4141 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4201 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4261 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4321 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4381 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4441 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4501 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4561 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4621 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4681 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4741 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4801 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4861 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4921 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4981 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5041 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5101 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5161 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5221 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5281 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5341 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5401 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5461 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5521 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5581 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5641 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5701 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5761 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5821 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5881 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5941 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6001 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6061 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6121 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6181 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     6241 acgaggccct ttcgtc
//
