LOCUS       sequence-401865-        7684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast integrative vector for the expression of the Sapphire-tagged 
            nuclear localizing 40nm nucGEMs under the INO4 promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7684)
  AUTHORS   Shu T et al.
  TITLE     nucGEMs probe the biophysical properties of the nucleoplasm
  JOURNAL   bioRxiv 2022
REFERENCE   2  (bases 1 to 7684)
  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-pINO4-SV40NLS-PfV-GS-Sapphire
FEATURES             Location/Qualifiers
     source          1..7684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(34..56)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     156..175
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(669..1763)
                     /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(1764..2171)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2472..2927)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2609..2630)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2821..2840
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3053..3075
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3067..3084
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3068..3084
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3091..3110
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3091..3109
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3300..3320
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4416..5132
                     /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      5153..5340
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5208..5232)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(5429..5447)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5468..5484)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5468..5484)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5481..5503)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5492..5508
                     /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(5516..5522,5523..5540,5541..5546))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5561..5582
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5699..5716)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5870..6458)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5950..5969)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6629..7420,7421..7489))
                     /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     7252..7271
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7490..7594)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7662..7680
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
       61 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
      121 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
      181 agtgcaccat atcgactacg tcgttaaggc cgtttctgac agagtaaaat tcttgaggga
      241 actttcacca ttatgggaaa tggttcaaga aggtattgac ttaaactcca tcaaatggtc
      301 aggtcattga gtgtttttta tttgttgtat tttttttttt ttagagaaaa tcctccaata
      361 tataaattag gaatcatagt ttcatgattt tctgttacac ctaacttttt gtgtggtgcc
      421 ctcctccttg tcaatattaa tgttaaagtg caattctttt tccttatcac gttgagccat
      481 tagtatcaat ttgcttacct gtattccttt acatcctcct ttttctcctt cttgataaat
      541 gtatgtagat tgcgtatata gtttcgtcta ccctatgaac atattccatt ttgtaatttc
      601 gtgtcgtttc tattatgaat ttcatttata aagtttatgt acaaatatca taaaaaaaga
      661 gaatcttttt aagcaaggat tttcttaact tcttcggcga cagcatcacc gacttcggtg
      721 gtactgttgg aaccacctaa atcaccagtt ctgatacctg catccaaaac ctttttaact
      781 gcatcttcaa tggccttacc ttcttcaggc aagttcaatg acaatttcaa catcattgca
      841 gcagacaaga tagtggcgat agggttgacc ttattctttg gcaaatctgg agcagaaccg
      901 tggcatggtt cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc caaggacgca
      961 gatggcaaca aacccaagga acctgggata acggaggctt catcggagat gatatcacca
     1021 aacatgttgc tggtgattat aataccattt aggtgggttg ggttcttaac taggatcatg
     1081 gcggcagaat caatcaattg atgttgaacc ttcaatgtag ggaattcgtt cttgatggtt
     1141 tcctccacag tttttctcca taatcttgaa gaggccaaaa cattagcttt atccaaggac
     1201 caaataggca atggtggctc atgttgtagg gccatgaaag cggccattct tgtgattctt
     1261 tgcacttctg gaacggtgta ttgttcacta tcccaagcga caccatcacc atcgtcttcc
     1321 tttctcttac caaagtaaat acctcccact aattctctga caacaacgaa gtcagtacct
     1381 ttagcaaatt gtggcttgat tggagataag tctaaaagag agtcggatgc aaagttacat
     1441 ggtcttaagt tggcgtacaa ttgaagttct ttacggattt ttagtaaacc ttgttcaggt
     1501 ctaacactac cggtacccca tttaggacca cccacagcac ctaacaaaac ggcatcagcc
     1561 ttcttggagg cttccagcgc ctcatctgga agtggaacac ctgtagcatc gatagcagca
     1621 ccaccaatta aatgattttc gaaatcgaac ttgacattgg aacgaacatc agaaatagct
     1681 ttaagaacct taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc tggcaaaacg
     1741 acgatcttct taggggcaga cattagaatg gtatatcctt gaaatatata tatatatatt
     1801 gctgaaatgt aaaaggtaag aaaagttaga aagtaagacg attgctaacc acctattgga
     1861 aaaaacaata ggtccttaaa taatattgtc aacttcaagt attgtgatgc aagcatttag
     1921 tcatgaacgc ttctctattc tatatgaaaa gccggttccg gcgctctcac ctttcctttt
     1981 tctcccaatt tttcagttga aaaaggtata tgcgtcaggc gacctctgaa attaacaaaa
     2041 aatttccagt catcgaattt gattctgtgc gatagcgccc ctgtgtgttc tcgttatgtt
     2101 gaggaaaaaa ataatggttg ctaagagatt cgaactcttg catcttacga tacctgagta
     2161 ttcccacagt taactgcggt caagatattt cttgaatcag gcgccttaga ccgctcggcc
     2221 aaacaaccaa ttacttgttg agaaatagag tataattatc ctataaatat aacgtttttg
     2281 aacacacatg aacaaggaag tacaggacaa ttgattttga agagaatgtg gattttgatg
     2341 taattgttgg gattccattt ttaataaggc aataatatta ggtatgtaga tatactagaa
     2401 gttctcctcg accggtcgat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     2461 accgcatcag gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     2521 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2581 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2641 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2701 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2761 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2821 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2881 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcca ttcgccattc
     2941 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     3001 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
     3061 cgacgttgta aaacgacggc cagtgaattg taatacgact cactataggg cgaattggag
     3121 ctctcttggc ttggtttgta tgagcttgtt acgtatcaaa caaaatattc acatgttttt
     3181 ctcaccttaa agacagaggc gcgattgcgt ttgcgaaatt ccaatgccaa caagataagc
     3241 ttgacgaagg agttaagagg gcggcttgaa ctaaaaagag aaaagcaata tctagaatgc
     3301 caaaaaagaa gagaaaggtc ctctcaataa atccaaccct tataaatagg gataagccat
     3361 acaccaaaga agaattaatg gaaattctga gattagcaat aatagctgag cttgatgcaa
     3421 taaatctata tgagcagatg gcaagatatt ccgaagatga aaacgtaaga aagattctcc
     3481 ttgatgttgc aagggaggag aaggctcacg ttggagaatt catggcactc cttctaaatt
     3541 tggatccaga acaagtaact gagctgaagg gtggatttga ggaagtcaag gagctaactg
     3601 gaattgaggc tcacataaat gataacaaga aggaggaatc taacgtggaa tattttgaga
     3661 agttgagaag tgctcttttg gatggggtaa ataagggaag atctttacta aagcacttgc
     3721 cagtaacgag aatagaagga caatcattca gggttgacat cattaaattt gaagatggtg
     3781 ttagggtagt aaagcaagag tacaagccaa ttccattatt gaagaagaaa ttctatgtgg
     3841 gaataagaga actcaacgat gggacttacg atgtatctat agcaactaaa gctggggaac
     3901 tcctagttaa agacgaagag tcgctagtaa taagagaaat tctatccaca gagggaatta
     3961 agaaaatgaa actttcctcc tgggacaatc cagaagaagc acttaatgat ttaatgaacg
     4021 cactgcagga agcctcaaat gcgtcagctg gaccatttgg gctgataatt aacccaaaaa
     4081 gatatgcaaa gttgctaaaa atttacgaga agagtggaaa gatgctagtt gaagtgctaa
     4141 aagagatatt caggggagga atcattgtta ctctaaacat tgatgagaat aaggtaataa
     4201 tatttgccaa tacaccagct gttcttgacg ttgtagttgg ccaagatgtg actctacaag
     4261 agctaggacc tgaaggagac gacgttgcat tcttggttag tgaagctata ggaataagaa
     4321 taaagaaccc agaggcaata gtagtcttag agtggtctgg cagcggtagt gggtcgggat
     4381 caggctccgg tcgacggatc cccgggttaa ttaagatgtc taaaggtgaa gaattattca
     4441 ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac aaattttctg
     4501 tctccggtga aggtgaaggt gatgctactt acggtaaatt gaccttaaaa tttatttgta
     4561 ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttttct tatggtgttc
     4621 aatgttttgc tagataccca gatcatatga aacaacatga ctttttcaag tctgccatgc
     4681 cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac tacaagacca
     4741 gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta aaaggtattg
     4801 attttaaaga agatggtaac attttaggtc acaaattgga atacaacttt aactctcaca
     4861 atgtttacat catggctgac aaacaaaaga atggtatcaa agttaacttc aaaattagac
     4921 acaacattga agatggttct gttcaattag ctgaccatta tcaacaaaat actccaattg
     4981 gtgatggtcc agtcttgtta ccagacaacc attacttatc cattcaatct gccttatcca
     5041 aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttgttact gctgctggta
     5101 ttacccatgg tattgatgaa ttgtacaaat aaggcgcgcc acttctaaat aagcgaattt
     5161 cttatgattt atgattttta ttattaaata agttataaaa aaaataagtg tatacaaatt
     5221 ttaaagtgac tcttaggttt taaaacgaaa attcttattc ttgagtaact ctttcctgta
     5281 ggtcaggttg ctttctcagg tatagtatga ggtcgctctt attgaccaca cctctaccgg
     5341 cagatccgct agggataaca gggtaatata gatctgttta gcttgcctcg tccccgctcg
     5401 agggggggcc cggtaccagc ttttgttccc tttagtgagg gttaatttcg agcttggcgt
     5461 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5521 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5581 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5641 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5701 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5761 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5821 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5881 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5941 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6001 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6061 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6121 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6181 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6241 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6301 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6361 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6421 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6481 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6541 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6601 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6661 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6721 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6781 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6841 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6901 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6961 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7021 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7081 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7141 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7201 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7261 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7321 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7381 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7441 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7501 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7561 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7621 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     7681 cctt
//
