LOCUS       sequence-131819-        7268 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Uracil selection; Expression of PP7-2x-yeGFP fusion protein under 
            the MET25 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7268)
  AUTHORS   Saroufim MA, Bensidoun P, Raymond P, Rahman S, Krause MR, Oeffinger 
            M, Zenklusen D
  TITLE     The nuclear basket mediates perinuclear mRNA scanning in budding 
            yeast.
  JOURNAL   J Cell Biol. 2015 Dec 21;211(6):1131-40. doi: 10.1083/jcb.201503070.
  PUBMED    26694838
REFERENCE   2  (bases 1 to 7268)
  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: "10.1083/jcb"; 
            journalName: "J Cell Biol"; date: "2015-12-21- 21"; volume: "211"; 
            issue: "6"; pages: "1131-40"
COMMENT     Sequence Label: pDZ514 pURA MET25p PP7-PS-2x-yeGFP
FEATURES             Location/Qualifiers
     source          1..7268
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        34..383
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     CDS             789..1499
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             1509..2222
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     terminator      2232..2479
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2248..2266)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(2501..2519)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2540..2556)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2540..2556)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2553..2575)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2564..2580
                     /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(2588..2594,2595..2612,2613..2618))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2633..2654
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2771..2788)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2942..3530)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3022..3041)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3701..4492,4493..4561))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4324..4343
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4562..4666)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    4703..5206
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     5248..5266
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5304..5326)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5426..5445
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5465..5685
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             5686..6489
                     /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(6618..7073)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6755..6776)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6967..6986
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7199..7221
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7213..7230
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7214..7230
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7237..7256
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7237..7255
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ccggatgcaa gggttcgaat cccttagctc tcattatttt ttgctttttc tcttgaggtc
       61 acatgatcgc aaaatggcaa atggcacgtg aagctgtcga tattggggaa ctgtggtggt
      121 tggcaaatga ctaattaagt tagtcaaggc gccatcctca tgaaaactgt gtaacataat
      181 aaccgaagtg tcgaaaaggt ggcaccttgt ccaattgaac acgctcgatg aaaaaaataa
      241 gatatatata aggttaagta aagcgtctgt tagaaaggaa gtttttcctt tttcttgctc
      301 tcttgtcttt tcatctacta tttccttcgt gtaatacagg gtcgtcagat acatagatac
      361 aattctatta cccccatcca tactctagaa tgtccaaaac catcgttctt tcggtcggcg
      421 aggctactcg cactctgact gagatccagt ccaccgcaga ccgtcagatc ttcgaagaga
      481 aggtcgggcc tctggtgggt cggctgcgcc tcacggcttc gctccgtcaa aacggagcca
      541 agaccgcgta tcgcgtcaac ctaaaactgg atcaggcgga cgtcgttgat tccggacttc
      601 cgaaagtgcg ctacactcag gtatggtcgc acgacgtgac aatcgttgcg aatagcaccg
      661 aggcctcgcg caaatcgttg tacgatttga ccaagtccct cgtcgcgacc tcgcaggtcg
      721 aagatcttgt cgtcaacctt gtgccgctgg gccgtggatc ccccgggctg caggaattcg
      781 atatcgtgtc taaaggtgaa gaattattca ctggtgttgt cccaattttg gttgaattag
      841 atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt gatgctactt
      901 acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt ccatggccaa
      961 ccttagtcac tactttcggt tatggtgttc aatgttttgc gagataccca gatcatatga
     1021 aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa agaactattt
     1081 ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa ggtgatacct
     1141 tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac attttaggtc
     1201 acaaattgga atacaactat aactctcaca atgtttacat catggctgac aaacaaaaga
     1261 atggtatcaa agttaacttc aaaattagac acaacattga agatggttct gttcaattag
     1321 ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta ccagacaacc
     1381 attacttatc cactcaatct gccttatcca aagatccaaa cgaaaagaga gaccacatgg
     1441 tcttgttaga atttgttact gctgctggta ttacccatgg tatggatgaa ttgtacaaag
     1501 atatcgtgtc taaaggtgaa gaattattca ctggtgttgt cccaattttg gttgaattag
     1561 atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt gatgctactt
     1621 acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt ccatggccaa
     1681 ccttagtcac tactttcggt tatggtgttc aatgttttgc gagataccca gatcatatga
     1741 aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa agaactattt
     1801 ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa ggtgatacct
     1861 tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac attttaggtc
     1921 acaaattgga atacaactat aactctcaca atgtttacat catggctgac aaacaaaaga
     1981 atggtatcaa agttaacttc aaaattagac acaacattga agatggttct gttcaattag
     2041 ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta ccagacaacc
     2101 attacttatc cactcaatct gccttatcca aagatccaaa cgaaaagaga gaccacatgg
     2161 tcttgttaga atttgttact gctgctggta ttacccatgg tatggatgaa ttgtacaaat
     2221 aactggtcga gtcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc
     2281 cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt
     2341 tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta
     2401 cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg
     2461 ctcgaaggct ttaatttgcg gccggtacca gcttttgttc cctttagtga gggttaattt
     2521 cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     2581 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     2641 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2701 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2761 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2821 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2881 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2941 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3001 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3061 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3121 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3181 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3241 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3301 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3361 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     3421 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3481 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3541 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3601 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3661 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3721 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctgcccgtcg
     3781 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3841 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3901 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3961 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4021 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4081 caaggcgagt tacatgatcc cccatgttgt gaaaaaaagc ggttagctcc ttcggtcctc
     4141 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4201 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4261 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4321 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4381 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4441 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4501 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4561 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4621 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4681 aagtgccacc tgggtccttt tcatcacgtg ctataaaaat aattataatt taaatttttt
     4741 aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa aaaatagttt
     4801 ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta ccttttatct
     4861 tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt gtagaagacc
     4921 acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt atatctattt
     4981 aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt gaaatttttt
     5041 aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt tatttacttt
     5101 ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc caaattattc
     5161 catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatccgtc ctaagaaacc
     5221 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     5281 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     5341 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     5401 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccac
     5461 gcttttcaat tcaattcatc attttttttt tattcttttt tttgatttcg gtttctttga
     5521 aatttttttg attcggtaat ctccgaacag aaggaagaac gaaggaagga gcacagactt
     5581 agattggtat atatacgcat atgtagtgtt gaagaaacat gaaattgccc agtattctta
     5641 acccaactgc acagaacaaa aacctgcagg aaacgaagat aaatcatgtc gaaagctaca
     5701 tataaggaac gtgctgctac tcatcctagt cctgttgctg ccaagctatt taatatcatg
     5761 cacgaaaagc aaacaaactt gtgtgcttca ttggatgttc gtaccaccaa ggaattactg
     5821 gagttagttg aagcattagg tcccaaaatt tgtttactaa aaacacatgt ggatatcttg
     5881 actgattttt ccatggaggg cacagttaag ccgctaaagg cattatccgc caagtacaat
     5941 tttttactct tcgaagacag aaaatttgct gacattggta atacagtcaa attgcagtac
     6001 tctgcgggtg tatacagaat agcagaatgg gcagacatta cgaatgcaca cggtgtggtg
     6061 ggcccaggta ttgttagcgg tttgaagcag gcggcagaag aagtaacaaa ggaacctaga
     6121 ggccttttga tgttagcaga attgtcatgc aagggctccc tatctactgg agaatatact
     6181 aagggtactg ttgacattgc gaagagcgac aaagattttg ttatcggctt tattgctcaa
     6241 agagacatgg gtggaagaga tgaaggttac gattggttga ttatgacacc cggtgtgggt
     6301 ttagatgaca agggagacgc attgggtcaa cagtatagaa ccgtggatga tgtggtctct
     6361 acaggatctg acattattat tgttggaaga ggactatttg caaagggaag ggatgctaag
     6421 gtagagggtg aacgttacag aaaagcaggc tgggaagcat atttgagaag atgcggccag
     6481 caaaactaaa aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct
     6541 tcaatttaat tatatcagtt attaccctgc ggtgtgaaat accgcacaga tgcgtaagga
     6601 gaaaataccg catcaggaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt
     6661 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     6721 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     6781 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     6841 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     6901 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     6961 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     7021 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtccattcg
     7081 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     7141 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     7201 cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa
     7261 ttggagct
//
