LOCUS       sequence-368135-        7571 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GAL::Rfa1[330-621]-GFP under control of GAL1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7571)
  AUTHORS   Belanger KD, Griffith AL, Baker HL, Hansen JN, Kovacs LA, Seconi JS,
            Strine AC
  TITLE     The karyopherin Kap95 and the C-termini of Rfa1, Rfa2, and Rfa3 are 
            necessary for efficient nuclear import of functional RPA complex 
            proteins in Saccharomyces cerevisiae.
  JOURNAL   DNA Cell Biol. 2011 Sep;30(9):641-51. doi: 10.1089/dna.2010.1071. 
            Epub 2011 Feb 20.
  PUBMED    21332387
REFERENCE   2  (bases 1 to 7571)
  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: 
            "10.1089/dna.2010.1071"; journalName: "DNA Cell Biol"; date: 
            "2011-09"; volume: "30"; issue: "9"; pages: "641-51"
COMMENT     Sequence Label: KBB372
FEATURES             Location/Qualifiers
     source          1..7571
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     124..143
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     377..394
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    511..532
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(547..552,553..570,571..577)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    585..601
                     /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     590..612
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     609..625
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     609..625
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     644..664
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        646..664
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1073..1096
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             1993..2703
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2020..2048)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2643..2663
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2754..2772)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2768..2815
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3193..3209)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3194..3210)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      3212..3459
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3228..3246)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3477..3496)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3478..3496)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3506..3523)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3506..3522)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3515..3537)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3663..4118
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3750..3769)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3960..3981
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4249..5052)
                     /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"
     promoter        complement(5053..5268)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5292..5311)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5411..5433
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5471..5489)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5530..6033
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6070..6174
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6175..6243,6244..7035)
                     /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(6393..6412)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7206..7571,1..223)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
       61 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
      121 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
      181 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
      241 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
      301 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
      361 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
      421 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
      481 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
      541 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
      601 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
      661 aagggaacaa aagctggagc tcgtgacagc cctccgaagg aagactctcc tccgtgcgtc
      721 ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca ctgctccgaa
      781 caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt ggcagtaacc
      841 tggccccaca aaccttcaaa tgaacgaatc aaattaacaa ccataggatg ataatgcgat
      901 tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt ttgatctatt
      961 aacggatata taaatgcaaa aactgcataa ccactttaac taatactttc aacattttcg
     1021 gtttgtatta cttcttattc aaatgtaata aaagtatcaa caaaaaattg ttaatatacc
     1081 tctatacttt aacgtcaagg agaaaaaact ataatgtttg agctaacttc aagggctggg
     1141 aagaaattcg atcgtcgtga catcacaatt gttgacgact ctgggttttc tatctctgtt
     1201 ggcctatgga atcagcaagc ccttgatttc aaccttcctg aaggttctgt tgctgccatt
     1261 aaaggtgttc gtgtgacgga ttttggtggc aaatctttgt ctatgggatt ttctagtacc
     1321 ctgattccga atccagaaat tcctgaggca tatgccttaa agggttggta tgattccaag
     1381 ggccgcaacg caaacttcat cactttaaag caagaacccg gtatgggtgg tcaatcggct
     1441 gctagcttaa caaaattcat tgctcagcgt attactattg ctagagctca agctgaaaat
     1501 ctaggaagaa gcgagaaagg tgactttttt agtgttaaag ctgctataag tttcttaaaa
     1561 gttgataatt ttgcatatcc tgcctgttct aatgagaatt gtaataagaa agttctggaa
     1621 cagcctgatg gtacttggag atgtgagaag tgcgacacca ataatgcaag gccaaattgg
     1681 agatacatct tgacaatatc aattattgac gaaaccaatc aactatggct cactttattt
     1741 gacgaccaag ctaaacaatt attgggtgtt gatgctaata cattaatgtc tttgaaggaa
     1801 gaagacccca acgaattcac aaaaattact caaagtatcc aaatgaacga atatgacttt
     1861 aggattagag cgcgtgagga tacatacaat gatcaaagca gaattagata taccgttgct
     1921 aacctacaca gcttgaatta cagggctgaa gccgactatc ttgccgatga gttatccaag
     1981 gctttgttag ctagcaaagg agaagaactc ttcactggag ttgtcccaat tcttgttgaa
     2041 ttagatggtg atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca
     2101 acatacggaa aacttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg
     2161 ccaacacttg tcactacttt cacttatggt gttcagtgct tttcaagata cccggatcat
     2221 atgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaaagaact
     2281 atatttttca aagatgacgg gaactacaag acacgtgctg aagtcaagtt tgaaggtgat
     2341 acccttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg aaacattctt
     2401 gggcacaaat tggaatacaa ctataactca cacaatgtat acatcatggc agacaaacaa
     2461 aagaatggaa tcaaagctaa cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa
     2521 ctagcagacc attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac
     2581 aaccattacc tgtccacaca atctgccctt tcgaaagatc ccaacgaaaa gagagaccac
     2641 atggtccttc ttgagtttgt aacagctgct gggattacac atggcatgga tgaactatac
     2701 aaaggatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     2761 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     2821 aggaggaact atatccggat atccacagga cgggtgtggt cgccatgatc gcgtagtcga
     2881 tagtggctcc aagtagcgaa gcgagcagga ctgggcggcg gccaaagcgg tcggacagtg
     2941 ctccgagaac gggtgcgcat agaaattgca tcaacgcata tagcgctagc agcacgccat
     3001 agtgactggc gatgctgtcg gaatggacga tatcccgcaa gaggcccggc agtaccggca
     3061 taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg atgagcgcat
     3121 tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc
     3181 attaaagctt atcgataccg tcgacctcga gtcatgtaat tagttatgtc acgcttacat
     3241 tcacgccctc cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc
     3301 taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa
     3361 atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg
     3421 cttgagaagg ttttgggacg ctcgaaggct ttaatttgcg gccggtaccc aattcgccct
     3481 atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     3541 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     3601 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     3661 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3721 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3781 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3841 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3901 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3961 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     4021 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     4081 taacgcgaat tttaacaaaa tattaacgct tacaatttcc tgatgcggta ttttctcctt
     4141 acgcatctgt gcggtatttc acaccgcata gggtaataac tgatataatt aaattgaagc
     4201 tctaatttgt gagtttagta tacatgcatt tacttataat acagtttttt agttttgctg
     4261 gccgcatctt ctcaaatatg cttcccagcc tgcttttctg taacgttcac cctctacctt
     4321 agcatccctt ccctttgcaa atagtcctct tccaacaata ataatgtcag atcctgtaga
     4381 gaccacatca tccacggttc tatactgttg acccaatgcg tctcccttgt catctaaacc
     4441 cacaccgggt gtcataatca accaatcgta accttcatct cttccaccca tgtctctttg
     4501 agcaataaag ccgataacaa aatctttgtc gctcttcgca atgtcaacag tacccttagt
     4561 atattctcca gtagataggg agcccttgca tgacaattct gctaacatca aaaggcctct
     4621 aggttccttt gttacttctt ctgccgcctg cttcaaaccg ctaacaatac ctgggcccac
     4681 cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt ctgtatacac ccgcagagta
     4741 ctgcaatttg actgtattac caatgtcagc aaattttctg tcttcgaaga gtaaaaaatt
     4801 gtacttggcg gataatgcct ttagcggctt aactgtgccc tccatggaaa aatcagtcaa
     4861 gatatccaca tgtgttttta gtaaacaaat tttgggacct aatgcttcaa ctaactccag
     4921 taattccttg gtggtacgaa catccaatga agcacacaag tttgtttgct tttcgtgcat
     4981 gatattaaat agcttggcag caacaggact aggatgagta gcagcacgtt ccttatatgt
     5041 agctttcgac atgatttatc ttcgtttcct gcaggttttt gttctgtgca gttgggttaa
     5101 gaatactggg caatttcatg tttcttcaac actacatatg cgtatatata ccaatctaag
     5161 tctgtgctcc ttccttcgtt cttccttctg ttcggagatt accgaatcaa aaaaatttca
     5221 aggaaaccga aatcaaaaaa aagaataaaa aaaaaatgat gaattgaaaa ggtggtatgg
     5281 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     5341 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5401 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5461 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     5521 tcttagacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata
     5581 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt
     5641 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa
     5701 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata
     5761 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct
     5821 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata
     5881 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact
     5941 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa
     6001 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg
     6061 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     6121 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     6181 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6241 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6301 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6361 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6421 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6481 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6541 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6601 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6661 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6721 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6781 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6841 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6901 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6961 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     7021 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     7081 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     7141 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     7201 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     7261 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     7321 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     7381 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7441 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7501 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7561 acgacctaca c
//
