LOCUS       sequence-232728-        6028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast integrative vector for the exression of the 20nm GEM under the
            HIS3 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6028)
  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 6028)
  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-URA3-PHIS3-AqLumSynth –Sapphire
FEATURES             Location/Qualifiers
     source          1..6028
                     /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             2766..3482
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSIQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGIDELYK"
     promoter        complement(3768..3786)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3807..3823)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3807..3823)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3820..3842)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3831..3847
                     /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(3855..3861,3862..3879,3880..3885))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3900..3921
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4038..4055)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4209..4797)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4289..4308)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4968..5759,5760..5828))
                     /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     5591..5610
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5829..5933)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6001..6019
                     /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 agtatgcaaa tatacgaagg caagctgact gcggaaggct
     2281 taagattcgg cattgtagct agtaggttta accatgctct ggtcgatagg ctagttgaag
     2341 gagcaattga ctgtatcgtg cgtcatggtg gtagagaaga ggatatcacg ctagtcaggg
     2401 ttcccggttc ttgggaaatc cctgtggctg ctggggaatt ggctaggaaa gaggacattg
     2461 acgctgtaat cgccattggt gtacttatta gaggagcaac acctcatttc gattatatag
     2521 caagtgaagt cagcaaagga ctagctaatt tgtctttgga gcttagaaaa cctatcactt
     2581 tcggtgttat cacagctgac accttagaac aagctataga acgtgctgga actaaacatg
     2641 gtaataaagg atgggaagct gctctgtccg ccattgagat ggcaaatttg tttaagagct
     2701 tgagatctgg cagcggtagt gggtcgggat caggctccgg tcgacggatc cccgggttaa
     2761 ttaagatgtc taaaggtgaa gaattattca ctggtgttgt cccaattttg gttgaattag
     2821 atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt gatgctactt
     2881 acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt ccatggccaa
     2941 ccttagtcac tactttttct tatggtgttc aatgttttgc tagataccca gatcatatga
     3001 aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa agaactattt
     3061 ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa ggtgatacct
     3121 tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac attttaggtc
     3181 acaaattgga atacaacttt aactctcaca atgtttacat catggctgac aaacaaaaga
     3241 atggtatcaa agttaacttc aaaattagac acaacattga agatggttct gttcaattag
     3301 ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta ccagacaacc
     3361 attacttatc cattcaatct gccttatcca aagatccaaa cgaaaagaga gaccacatgg
     3421 tcttgttaga atttgttact gctgctggta ttacccatgg tattgatgaa ttgtacaaat
     3481 aaggcgcgcc attataagta aatgcatgta tactaaactc acaaattaga gcttcaattt
     3541 aattatatca gttattaccc gggaatctcg gtcgtaatga tttctataat gacgaaaaaa
     3601 aaaaaattgg aaagaaaaag cttcatggcc tttataaaaa ggaactatcc aatacctcgc
     3661 cagaaccaag taacagtatt ttacggggca caaatcaaga acaataagac aggactgtaa
     3721 agatggacgc atagcctcga gggggggccc ggtaccagct tttgttccct ttagtgaggg
     3781 ttaatttcga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3841 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3901 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3961 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4021 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4081 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4141 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4201 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4261 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4321 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4381 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4441 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4501 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4561 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4621 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4681 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4741 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4801 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4861 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4921 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4981 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5041 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5101 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5161 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5221 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5281 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5341 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5401 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5461 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5521 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5581 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5641 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5701 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5761 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5821 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5881 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5941 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     6001 aataggcgta tcacgaggcc ctttcgtc
//
