LOCUS       sequence-402536-        6648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast integrative vector for the expression of the Sapphire-tagged 
            nuclear localizing 40nm GEMs under the HIS3 promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6648)
  AUTHORS   Shu T et al.
  TITLE     nucGEMs probe the biophysical properties of the nucleoplasm
  JOURNAL   bioRxiv 2022
REFERENCE   2  (bases 1 to 6648)
  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: pRS403-HIS3-pINO4-SV40NLS-PfV-GS-Sapphire
FEATURES             Location/Qualifiers
     source          1..6648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     386..403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    520..541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(556..561,562..579,580..586)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    594..610
                     /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     599..621
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     618..634
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     618..634
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     653..673
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        655..673
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             868..888
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1984..2700
                     /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      2721..2908
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2776..2800)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2993..3012)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2994..3012)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3022..3039)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3022..3038)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3031..3053)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3179..3634
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3266..3285)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3476..3497
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3894..4556)
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        complement(4557..4744)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     primer_bind     complement(4891..4910)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5010..5032
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5070..5088)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5156..5260
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5261..5329,5330..6121)
                     /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(5479..5498)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6292..6648,1..232)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
       61 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
      421 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
      481 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
      541 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
      601 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
      661 ccctcactaa agggaacaaa agctggagct ctcttggctt ggtttgtatg agcttgttac
      721 gtatcaaaca aaatattcac atgtttttct caccttaaag acagaggcgc gattgcgttt
      781 gcgaaattcc aatgccaaca agataagctt gacgaaggag ttaagagggc ggcttgaact
      841 aaaaagagaa aagcaatatc tagaatgcca aaaaagaaga gaaaggtcct ctcaataaat
      901 ccaaccctta taaataggga taagccatac accaaagaag aattaatgga aattctgaga
      961 ttagcaataa tagctgagct tgatgcaata aatctatatg agcagatggc aagatattcc
     1021 gaagatgaaa acgtaagaaa gattctcctt gatgttgcaa gggaggagaa ggctcacgtt
     1081 ggagaattca tggcactcct tctaaatttg gatccagaac aagtaactga gctgaagggt
     1141 ggatttgagg aagtcaagga gctaactgga attgaggctc acataaatga taacaagaag
     1201 gaggaatcta acgtggaata ttttgagaag ttgagaagtg ctcttttgga tggggtaaat
     1261 aagggaagat ctttactaaa gcacttgcca gtaacgagaa tagaaggaca atcattcagg
     1321 gttgacatca ttaaatttga agatggtgtt agggtagtaa agcaagagta caagccaatt
     1381 ccattattga agaagaaatt ctatgtggga ataagagaac tcaacgatgg gacttacgat
     1441 gtatctatag caactaaagc tggggaactc ctagttaaag acgaagagtc gctagtaata
     1501 agagaaattc tatccacaga gggaattaag aaaatgaaac tttcctcctg ggacaatcca
     1561 gaagaagcac ttaatgattt aatgaacgca ctgcaggaag cctcaaatgc gtcagctgga
     1621 ccatttgggc tgataattaa cccaaaaaga tatgcaaagt tgctaaaaat ttacgagaag
     1681 agtggaaaga tgctagttga agtgctaaaa gagatattca ggggaggaat cattgttact
     1741 ctaaacattg atgagaataa ggtaataata tttgccaata caccagctgt tcttgacgtt
     1801 gtagttggcc aagatgtgac tctacaagag ctaggacctg aaggagacga cgttgcattc
     1861 ttggttagtg aagctatagg aataagaata aagaacccag aggcaatagt agtcttagag
     1921 tggtctggca gcggtagtgg gtcgggatca ggctccggtc gacggatccc cgggttaatt
     1981 aagatgtcta aaggtgaaga attattcact ggtgttgtcc caattttggt tgaattagat
     2041 ggtgatgtta atggtcacaa attttctgtc tccggtgaag gtgaaggtga tgctacttac
     2101 ggtaaattga ccttaaaatt tatttgtact actggtaaat tgccagttcc atggccaacc
     2161 ttagtcacta ctttttctta tggtgttcaa tgttttgcta gatacccaga tcatatgaaa
     2221 caacatgact ttttcaagtc tgccatgcca gaaggttatg ttcaagaaag aactattttt
     2281 ttcaaagatg acggtaacta caagaccaga gctgaagtca agtttgaagg tgatacctta
     2341 gttaatagaa tcgaattaaa aggtattgat tttaaagaag atggtaacat tttaggtcac
     2401 aaattggaat acaactttaa ctctcacaat gtttacatca tggctgacaa acaaaagaat
     2461 ggtatcaaag ttaacttcaa aattagacac aacattgaag atggttctgt tcaattagct
     2521 gaccattatc aacaaaatac tccaattggt gatggtccag tcttgttacc agacaaccat
     2581 tacttatcca ttcaatctgc cttatccaaa gatccaaacg aaaagagaga ccacatggtc
     2641 ttgttagaat ttgttactgc tgctggtatt acccatggta ttgatgaatt gtacaaataa
     2701 ggcgcgccac ttctaaataa gcgaatttct tatgatttat gatttttatt attaaataag
     2761 ttataaaaaa aataagtgta tacaaatttt aaagtgactc ttaggtttta aaacgaaaat
     2821 tcttattctt gagtaactct ttcctgtagg tcaggttgct ttctcaggta tagtatgagg
     2881 tcgctcttat tgaccacacc tctaccggca gatccgctag ggataacagg gtaatataga
     2941 tctgtttagc ttgcctcgtc cccgctcgag ggggggcccg gtacccaatt cgccctatag
     3001 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     3061 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     3121 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac
     3181 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3241 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3301 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3361 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     3421 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3481 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     3541 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     3601 gcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt ctccttacgc
     3661 atctgtgcgg tatttcacac cgcatagatc cgtcgagttc aagagaaaaa aaaagaaaaa
     3721 gcaaaaagaa aaaaggaaag cgcgcctcgt tcagaatgac acgtatagaa tgatgcatta
     3781 ccttgtcatc ttcagtatca tactgttcgt atacatactt actgacattc ataggtatac
     3841 atatatacac atgtatatat atcgtatgct gcagctttaa ataatcggtg tcactacata
     3901 agaacacctt tggtggaggg aacatcgttg gtaccattgg gcgaggtggc ttctcttatg
     3961 gcaaccgcaa gagccttgaa cgcactctca ctacggtgat gatcattctt gcctcgcaga
     4021 caatcaacgt ggagggtaat tctgctagcc tctgcaaagc tttcaagaaa atgcgggatc
     4081 atctcgcaag agagatctcc tactttctcc ctttgcaaac caagttcgac aactgcgtac
     4141 ggcctgttcg aaagatctac caccgctctg gaaagtgcct catccaaagg cgcaaatcct
     4201 gatccaaacc tttttactcc acgcacggcc cctagggcct ctttaaaagc ttgaccgaga
     4261 gcaatcccgc agtcttcagt ggtgtgatgg tcgtctatgt gtaagtcacc aatgcactca
     4321 acgattagcg accagccgga atgcttggcc agagcatgta tcatatggtc cagaaaccct
     4381 atacctgtgt ggacgttaat cacttgcgat tgtgtggcct gttctgctac tgcttctgcc
     4441 tctttttctg ggaagatcga gtgctctatc gctaggggac caccctttaa agagatcgca
     4501 atctgaatct tggtttcatt tgtaatacgc tttactaggg ctttctgctc tgtcatcttt
     4561 gccttcgttt atcttgcctg ctcatttttt agtatattct tcgaagaaat cacattactt
     4621 tatataatgt ataattcatt atgtgataat gccaatcgct aagaaaaaaa aagagtcatc
     4681 cgctaggtgg aaaaaaaaaa atgaaaatca ttaccgaggc ataaaaaaat atagagtgta
     4741 ctagaggagg ccaagagtaa tagaaaaaga aaattgcggg aaaggactgt gttatgactt
     4801 ccctgactaa tgccgtgttc aaacgatacc tggcagtgac tcctagcgct caccaagctc
     4861 ttaaaacggg aatttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4921 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4981 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5041 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     5101 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5161 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5221 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5281 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5341 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5401 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5461 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5521 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5581 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5641 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5701 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5761 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5821 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     5881 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5941 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6001 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6061 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6121 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6181 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6241 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6301 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6361 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6421 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6481 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6541 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6601 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaac
//
