LOCUS       sequence-401868-        7720 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast integrative vector for the expression of the GFPMUT3-tagged 
            nuclear localizing 40nm nucGEMs under the HIS3 promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7720)
  AUTHORS   Shu T et al.
  TITLE     nucGEMs probe the biophysical properties of the nucleoplasm
  JOURNAL   bioRxiv 2022
REFERENCE   2  (bases 1 to 7720)
  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: "bioRxiv 
            2022"
COMMENT     Sequence Label: pRS305-LEU2-pHIS3-SV40NLS-PfV-GS-GFPMUT3
FEATURES             Location/Qualifiers
     source          1..7720
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..46)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     146..165
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(659..1753)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1754..2161)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2462..2917)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2599..2620)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2811..2830
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3043..3065
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3057..3074
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3058..3074
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3081..3100
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3081..3099
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3131..3316
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             3326..3346
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4442..5158
                     /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"
     terminator      5179..5366
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5234..5258)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(5455..5473)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5494..5510)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5494..5510)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5507..5529)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5518..5534
                     /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(5542..5548,5549..5566,5567..5572))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5587..5608
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5725..5742)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5896..6484)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5976..5995)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6655..7446,7447..7515))
                     /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     7278..7297
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7516..7620)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7688..7706
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
       61 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
      121 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
      181 atcgactacg tcgttaaggc cgtttctgac agagtaaaat tcttgaggga actttcacca
      241 ttatgggaaa tggttcaaga aggtattgac ttaaactcca tcaaatggtc aggtcattga
      301 gtgtttttta tttgttgtat tttttttttt ttagagaaaa tcctccaata tataaattag
      361 gaatcatagt ttcatgattt tctgttacac ctaacttttt gtgtggtgcc ctcctccttg
      421 tcaatattaa tgttaaagtg caattctttt tccttatcac gttgagccat tagtatcaat
      481 ttgcttacct gtattccttt acatcctcct ttttctcctt cttgataaat gtatgtagat
      541 tgcgtatata gtttcgtcta ccctatgaac atattccatt ttgtaatttc gtgtcgtttc
      601 tattatgaat ttcatttata aagtttatgt acaaatatca taaaaaaaga gaatcttttt
      661 aagcaaggat tttcttaact tcttcggcga cagcatcacc gacttcggtg gtactgttgg
      721 aaccacctaa atcaccagtt ctgatacctg catccaaaac ctttttaact gcatcttcaa
      781 tggccttacc ttcttcaggc aagttcaatg acaatttcaa catcattgca gcagacaaga
      841 tagtggcgat agggttgacc ttattctttg gcaaatctgg agcagaaccg tggcatggtt
      901 cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc caaggacgca gatggcaaca
      961 aacccaagga acctgggata acggaggctt catcggagat gatatcacca aacatgttgc
     1021 tggtgattat aataccattt aggtgggttg ggttcttaac taggatcatg gcggcagaat
     1081 caatcaattg atgttgaacc ttcaatgtag ggaattcgtt cttgatggtt tcctccacag
     1141 tttttctcca taatcttgaa gaggccaaaa cattagcttt atccaaggac caaataggca
     1201 atggtggctc atgttgtagg gccatgaaag cggccattct tgtgattctt tgcacttctg
     1261 gaacggtgta ttgttcacta tcccaagcga caccatcacc atcgtcttcc tttctcttac
     1321 caaagtaaat acctcccact aattctctga caacaacgaa gtcagtacct ttagcaaatt
     1381 gtggcttgat tggagataag tctaaaagag agtcggatgc aaagttacat ggtcttaagt
     1441 tggcgtacaa ttgaagttct ttacggattt ttagtaaacc ttgttcaggt ctaacactac
     1501 cggtacccca tttaggacca cccacagcac ctaacaaaac ggcatcagcc ttcttggagg
     1561 cttccagcgc ctcatctgga agtggaacac ctgtagcatc gatagcagca ccaccaatta
     1621 aatgattttc gaaatcgaac ttgacattgg aacgaacatc agaaatagct ttaagaacct
     1681 taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc tggcaaaacg acgatcttct
     1741 taggggcaga cattagaatg gtatatcctt gaaatatata tatatatatt gctgaaatgt
     1801 aaaaggtaag aaaagttaga aagtaagacg attgctaacc acctattgga aaaaacaata
     1861 ggtccttaaa taatattgtc aacttcaagt attgtgatgc aagcatttag tcatgaacgc
     1921 ttctctattc tatatgaaaa gccggttccg gcgctctcac ctttcctttt tctcccaatt
     1981 tttcagttga aaaaggtata tgcgtcaggc gacctctgaa attaacaaaa aatttccagt
     2041 catcgaattt gattctgtgc gatagcgccc ctgtgtgttc tcgttatgtt gaggaaaaaa
     2101 ataatggttg ctaagagatt cgaactcttg catcttacga tacctgagta ttcccacagt
     2161 taactgcggt caagatattt cttgaatcag gcgccttaga ccgctcggcc aaacaaccaa
     2221 ttacttgttg agaaatagag tataattatc ctataaatat aacgtttttg aacacacatg
     2281 aacaaggaag tacaggacaa ttgattttga agagaatgtg gattttgatg taattgttgg
     2341 gattccattt ttaataaggc aataatatta ggtatgtaga tatactagaa gttctcctcg
     2401 accggtcgat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     2461 gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     2521 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     2581 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     2641 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     2701 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     2761 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     2821 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     2881 acacccgccg cgcttaatgc gccgctacag ggcgcgtcca ttcgccattc aggctgcgca
     2941 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     3001 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     3061 aaacgacggc cagtgaattg taatacgact cactataggg cgaattggag ctcattactc
     3121 ttggcctcct ctagtacact ctatattttt ttatgcctcg gtaatgattt tcgttttttt
     3181 tttccaccta gcggatgact cttttttttt cttagcgatt ggcattatca cataatgaat
     3241 tatacattat ataaagtaat gtgatttcta cgaagaatat actaaaaaat gagcaggcaa
     3301 gataaacgaa ggcaaatcta gaatgccaaa aaagaagaga aaggtcctct caataaatcc
     3361 aacccttata aatagggata agccatacac caaagaagaa ttaatggaaa ttctgagatt
     3421 agcaataata gctgagcttg atgcaataaa tctatatgag cagatggcaa gatattccga
     3481 agatgaaaac gtaagaaaga ttctccttga tgttgcaagg gaggagaagg ctcacgttgg
     3541 agaattcatg gcactccttc taaatttgga tccagaacaa gtaactgagc tgaagggtgg
     3601 atttgaggaa gtcaaggagc taactggaat tgaggctcac ataaatgata acaagaagga
     3661 ggaatctaac gtggaatatt ttgagaagtt gagaagtgct cttttggatg gggtaaataa
     3721 gggaagatct ttactaaagc acttgccagt aacgagaata gaaggacaat cattcagggt
     3781 tgacatcatt aaatttgaag atggtgttag ggtagtaaag caagagtaca agccaattcc
     3841 attattgaag aagaaattct atgtgggaat aagagaactc aacgatggga cttacgatgt
     3901 atctatagca actaaagctg gggaactcct agttaaagac gaagagtcgc tagtaataag
     3961 agaaattcta tccacagagg gaattaagaa aatgaaactt tcctcctggg acaatccaga
     4021 agaagcactt aatgatttaa tgaacgcact gcaggaagcc tcaaatgcgt cagctggacc
     4081 atttgggctg ataattaacc caaaaagata tgcaaagttg ctaaaaattt acgagaagag
     4141 tggaaagatg ctagttgaag tgctaaaaga gatattcagg ggaggaatca ttgttactct
     4201 aaacattgat gagaataagg taataatatt tgccaataca ccagctgttc ttgacgttgt
     4261 agttggccaa gatgtgactc tacaagagct aggacctgaa ggagacgacg ttgcattctt
     4321 ggttagtgaa gctataggaa taagaataaa gaacccagag gcaatagtag tcttagagtg
     4381 gtctggcagc ggtagtgggt cgggatcagg ctccggtcga cggatccccg ggttaattaa
     4441 gatgtctaaa ggtgaagaat tattcactgg tgttgtccca attttggttg aattagatgg
     4501 tgatgttaat ggtcacaaat tttctgtctc cggtgaaggt gaaggtgatg ctacttacgg
     4561 taaattgacc ttaaaattta tttgtactac tggtaaattg ccagttccat ggccaacctt
     4621 agtcactact ttttcttatg gtgttcaatg ttttgctaga tacccagatc atatgaaaca
     4681 acatgacttt ttcaagtctg ccatgccaga aggttatgtt caagaaagaa ctattttttt
     4741 caaagatgac ggtaactaca agaccagagc tgaagtcaag tttgaaggtg ataccttagt
     4801 taatagaatc gaattaaaag gtattgattt taaagaagat ggtaacattt taggtcacaa
     4861 attggaatac aactttaact ctcacaatgt ttacatcatg gctgacaaac aaaagaatgg
     4921 tatcaaagtt aacttcaaaa ttagacacaa cattgaagat ggttctgttc aattagctga
     4981 ccattatcaa caaaatactc caattggtga tggtccagtc ttgttaccag acaaccatta
     5041 cttatccatt caatctgcct tatccaaaga tccaaacgaa aagagagacc acatggtctt
     5101 gttagaattt gttactgctg ctggtattac ccatggtatt gatgaattgt acaaataagg
     5161 cgcgccactt ctaaataagc gaatttctta tgatttatga tttttattat taaataagtt
     5221 ataaaaaaaa taagtgtata caaattttaa agtgactctt aggttttaaa acgaaaattc
     5281 ttattcttga gtaactcttt cctgtaggtc aggttgcttt ctcaggtata gtatgaggtc
     5341 gctcttattg accacacctc taccggcaga tccgctaggg ataacagggt aatatagatc
     5401 tgtttagctt gcctcgtccc cgctcgaggg ggggcccggt accagctttt gttcccttta
     5461 gtgagggtta atttcgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5521 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5581 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5641 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5701 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5761 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5821 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5881 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5941 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     6001 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     6061 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     6121 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6181 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6241 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6301 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6361 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6421 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6481 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6541 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6601 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6661 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6721 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6781 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6841 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6901 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6961 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     7021 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     7081 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     7141 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     7201 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     7261 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     7321 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     7381 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7441 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7501 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7561 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7621 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     7681 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
//
