LOCUS       sequence-367444-        7107 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  rfa2D248 truncation fused to GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7107)
  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 7107)
  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: KBB349
FEATURES             Location/Qualifiers
     source          1..7107
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      813..1401
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1302..1321
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1555..1572
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1689..1710
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1725..1730,1731..1748,1749..1755)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1763..1779
                     /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     1768..1790
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1787..1803
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1787..1803
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1824..1842
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1870..1886
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1871..1887
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3165..3875
                     /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(3192..3220)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3815..3835
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3926..3944)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3940..3987
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4194..4213)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4195..4213)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4220..4237)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4220..4236)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4229..4251)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4377..4832
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4464..4483)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4674..4695
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4963..5766)
                     /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(5767..5982)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6006..6025)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6125..6147
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6185..6203)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    6244..6747
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6784..6888
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6889..6957,6958..7107,1..642)
                     /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(join(7107,1..19))
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
       61 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
      121 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
      181 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
      241 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
      301 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
      361 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
      421 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
      481 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
      541 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
      601 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
      661 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
      721 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
      781 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
      841 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
      901 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
      961 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     1021 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     1081 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     1141 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     1201 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     1261 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     1321 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     1381 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     1441 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     1501 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     1561 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     1621 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     1681 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     1741 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     1801 gaccatgatt acgccaagct cgaaattaac cctcactaaa gggaacaaaa gctggtaccg
     1861 ggccccccct cgaggtcgac ggtatcgata agcttgatat cgaattcctg caggcccatt
     1921 ttgaaaacct caacgttaag aaagtcattt agagcagcaa taacactaga tgtggatatt
     1981 tctcccgaaa tactaatttt tcccttaaat ttgtccacaa attgctgtac tagctggtat
     2041 gattcaccac tgatgatatg ttcagccttt ttcacatatg tctggtcgaa agaaccactt
     2101 ttcagagatt ctacttctgc agacagcaat aattttgcat gcaataacgt ctccataatt
     2161 ttgcaaccac tagtttactt tttgcctggt ctgatagctt ctttttagca attcctttgg
     2221 cctcgatgag cttccatttt caattttcat cttttttttc acgatgcgaa gtttcttaga
     2281 taattatcgc catatacgaa acgcgttagg aaacgcgttc tttcctttat aataagcaat
     2341 attatatcaa cttaaggtgg aagtaatgtg gcaaaatcga tagcgactat ctagaacagg
     2401 ctagtttaag catatacata atggcaagta tgtattagtg ctaggaattg gaggatttgc
     2461 tcttttctgc atcatttttt ttatttttat tggcgaatgt taaaattact aacaatggat
     2521 gggattccaa tacagcctat caaccatata acgaatattc atcagtaacg ggcggtggct
     2581 ttgagaactc tgagtcccgc ccaggtagtg gggagtcgga aactaacact agagttaaca
     2641 ccttgacacc tgtgacgatc aaacaaattc tagagtccaa acaggatatt caggacggcc
     2701 ccttcgtttc gcataaccaa gaacttcatc acgtttgttt tgtaggtgtg gtgagaaaca
     2761 ttacagacca tactgcaaat atttttttaa ctattgagga tggaactggt caaatagaag
     2821 tgagaaaatg gagcgaagat gcaaatgact tggctgccgg taacgatgac tcttctggta
     2881 aaggttatgg ttcgcaagtc gcccaacaat ttgaaattgg cggttacgta aaagtttttg
     2941 gttctttgaa agagtttggt ggtaagaaaa atatacagta tgcggtgatt aagcccatag
     3001 attcattcaa tgaagtgttg acgcatcact tggaagtcat caaatgtcat tccatagcca
     3061 gtggaatgat gaaacaacct ttggagagtg catccaacaa caatgggcaa tcattatttg
     3121 tcaaggatga taacgataca tcttccggct ccagtccgtt acaaagcaaa ggagaagaac
     3181 tcttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat
     3241 tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc cttaaattta
     3301 tttgcactac tggaaaacta cctgttccat ggccaacact tgtcactact ttcacttatg
     3361 gtgttcagtg cttttcaaga tacccggatc atatgaaacg gcatgacttt ttcaagagtg
     3421 ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac gggaactaca
     3481 agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc gagttaaaag
     3541 gtattgattt taaagaagat ggaaacattc ttgggcacaa attggaatac aactataact
     3601 cacacaatgt atacatcatg gcagacaaac aaaagaatgg aatcaaagct aacttcaaaa
     3661 ttagacacaa cattgaagat ggaagcgttc aactagcaga ccattatcaa caaaatactc
     3721 caattggcga tggccctgtc cttttaccag acaaccatta cctgtccaca caatctgccc
     3781 tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg
     3841 ctgggattac acatggcatg gatgaactat acaaaggatc cggctgctaa caaagcccga
     3901 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     3961 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatccacag
     4021 gacgggtgtg gtcgccatga tcgcgtagtc gatagtggct ccaagtagcg aagcgagcag
     4081 gactgggcgg cggccaaagc ggtcggacag tgctccgaga acgggtgcgc atagaaattg
     4141 catcaacgca tatagcgcta gagcggccgc caccgcggtg gagctccaat tcgccctata
     4201 gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     4261 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     4321 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggacgc
     4381 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4441 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4501 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4561 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     4621 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4681 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     4741 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4801 gaattttaac aaaatattaa cgcttacaat ttcctgatgc ggtattttct ccttacgcat
     4861 ctgtgcggta tttcacaccg catagggtaa taactgatat aattaaattg aagctctaat
     4921 ttgtgagttt agtatacatg catttactta taatacagtt ttttagtttt gctggccgca
     4981 tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt tcaccctcta ccttagcatc
     5041 ccttcccttt gcaaatagtc ctcttccaac aataataatg tcagatcctg tagagaccac
     5101 atcatccacg gttctatact gttgacccaa tgcgtctccc ttgtcatcta aacccacacc
     5161 gggtgtcata atcaaccaat cgtaaccttc atctcttcca cccatgtctc tttgagcaat
     5221 aaagccgata acaaaatctt tgtcgctctt cgcaatgtca acagtaccct tagtatattc
     5281 tccagtagat agggagccct tgcatgacaa ttctgctaac atcaaaaggc ctctaggttc
     5341 ctttgttact tcttctgccg cctgcttcaa accgctaaca atacctgggc ccaccacacc
     5401 gtgtgcattc gtaatgtctg cccattctgc tattctgtat acacccgcag agtactgcaa
     5461 tttgactgta ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa aattgtactt
     5521 ggcggataat gcctttagcg gcttaactgt gccctccatg gaaaaatcag tcaagatatc
     5581 cacatgtgtt tttagtaaac aaattttggg acctaatgct tcaactaact ccagtaattc
     5641 cttggtggta cgaacatcca atgaagcaca caagtttgtt tgcttttcgt gcatgatatt
     5701 aaatagcttg gcagcaacag gactaggatg agtagcagca cgttccttat atgtagcttt
     5761 cgacatgatt tatcttcgtt tcctgcaggt ttttgttctg tgcagttggg ttaagaatac
     5821 tgggcaattt catgtttctt caacactaca tatgcgtata tataccaatc taagtctgtg
     5881 ctccttcctt cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat ttcaaggaaa
     5941 ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg aaaaggtggt atggtgcact
     6001 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     6061 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     6121 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     6181 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     6241 acggatcgct tgcctgtaac ttacacgcgc ctcgtatctt ttaatgatgg aataatttgg
     6301 gaatttactc tgtgtttatt tatttttatg ttttgtattt ggattttaga aagtaaataa
     6361 agaaggtaga agagttacgg aatgaagaaa aaaaaataaa caaaggttta aaaaatttca
     6421 acaaaaagcg tactttacat atatatttat tagacaagaa aagcagatta aatagatata
     6481 cattcgatta acgataagta aaatgtaaaa tcacaggatt ttcgtgtgtg gtcttctaca
     6541 cagacaagat gaaacaattc ggcattaata cctgagagca ggaagagcaa gataaaaggt
     6601 agtatttgtt ggcgatcccc ctagagtctt ttacatcttc ggaaaacaaa aactattttt
     6661 tctttaattt ctttttttac tttctatttt taatttatat atttatatta aaaaatttaa
     6721 attataatta tttttatagc acgtgatgaa aaggacccag gtggcacttt tcggggaaat
     6781 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     6841 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     6901 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     6961 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     7021 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     7081 ccaatgatga gcacttttaa agttctg
//
