LOCUS       sequence-368001-        6738 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RFA3-GFP fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6738)
  AUTHORS   Belanger KD, Simmons LA, Roth JK, VanderPloeg KA, Lichten LB, 
            Fahrenkrog B
  TITLE     The karyopherin Msn5/Kap142 requires Nup82 for nuclear export and 
            performs a function distinct from translocation in RPA protein 
            import.
  JOURNAL   J Biol Chem. 2004 Oct 15;279(42):43530-9. doi: 
            10.1074/jbc.M407641200. Epub 2004 Aug 4.
  PUBMED    15294903
REFERENCE   2  (bases 1 to 6738)
  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: 
            "10.1074/jbc.M407641200"; journalName: "J Biol Chem"; date: 
            "2004-10-15- 15"; volume: "279"; issue: "42"; pages: "43530-9"
COMMENT     Sequence Label: KBB285
FEATURES             Location/Qualifiers
     source          1..6738
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    89..110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(125..130,131..148,149..155)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    163..179
                     /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     168..190
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     187..203
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     187..203
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        224..242
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     270..286
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     271..287
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1196..1906
                     /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(1223..1251)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1846..1866
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(1957..1975)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1971..2018
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2225..2244)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2226..2244)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2251..2268)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2251..2267)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2260..2282)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2408..2863
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2495..2514)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2705..2726
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2994..3797)
                     /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(3798..4013)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4037..4056)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4156..4178
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4216..4234)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4275..4778
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4815..4919
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4920..4988,4989..5780)
                     /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(5138..5157)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5951..6539
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6440..6459
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6693..6710
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
       61 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
      121 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
      181 ttcacacagg aaacagctat gaccatgatt acgccaagct cgaaattaac cctcactaaa
      241 gggaacaaaa gctggtaccg ggccccccct cgaggtcgac ggtatcgata agcttgatat
      301 cgaattcctg caggcaacgt caagattgat cctatgcttt cataatttcc ctacatgatg
      361 gcagtcatac tccaattaag tgctacaaga aagaggcctt cgcaacaact gtgtcaacgc
      421 ttgcaagtgc taaattgtac tacagcacca gtggtatctc ctatagtcag ttacatagta
      481 aaatattcag aggtccatta cataaatata tatatatatg tagaggcatc catccatgtt
      541 tgtcgatgca gctgcaagca cccaacaaga acagatgaca gccaacagtc gttgactaac
      601 gtaatgggcc actgctaaga agcatccacc gcccatgcca tggttggaat catgacaagt
      661 cgccctatat aatgccttcg ttctttaata ttgacgcgcc aaatgtggca atacgcgata
      721 tttttttcgt tcgtgtttaa ataaagagca atataacaca cccttcacca ttcttgtaga
      781 acatcgtcta ccagtaacac aagtaaaaat ggccagcgaa acaccaagag ttgaccccac
      841 agaaatctcc aacgtcaatg ctcctgtgtt taggataata gcgcaaatca aatcacaacc
      901 cactgaatct cagttgattt tacaatcgcc aaccatatca tcgaaaaacg gcagcgaggt
      961 tgaaatgatt acgttgaaca atattcgtgt atctatgaat aaaacatttg agatcgactc
     1021 gtggtatgag tttgtttgca ggaacaatga tgacggcgag ctaggatttt tgattctaga
     1081 cgctgtacta tgcaaattca aggagaacga agatttgagc ttaaacggtg tggttgcttt
     1141 acagagacta tgtaagaaat acccagaaat atacgctgct gctggatccg ctgctagcaa
     1201 aggagaagaa ctcttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa
     1261 tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac
     1321 ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac
     1381 tttcacttat ggtgttcagt gcttttcaag atacccggat catatgaaac ggcatgactt
     1441 tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga
     1501 cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat
     1561 cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttgggcaca aattggaata
     1621 caactataac tcacacaatg tatacatcat ggcagacaaa caaaagaatg gaatcaaagc
     1681 taacttcaaa attagacaca acattgaaga tggaagcgtt caactagcag accattatca
     1741 acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac
     1801 acaatctgcc ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt
     1861 tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaaggat ccggctgcta
     1921 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     1981 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     2041 gatatccaca ggacgggtgt ggtcgccatg atcgcgtagt cgatagtggc tccaagtagc
     2101 gaagcgagca ggactgggcg gcggccaaag cggtcggaca gtgctccgag aacgggtgcg
     2161 catagaaatt gcatcaacgc atatagcgct agagcggccg ccaccgcggt ggagctccaa
     2221 ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg
     2281 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     2341 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     2401 cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2461 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2521 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2581 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2641 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2701 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2761 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2821 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg cggtattttc
     2881 tccttacgca tctgtgcggt atttcacacc gcatagggta ataactgata taattaaatt
     2941 gaagctctaa tttgtgagtt tagtatacat gcatttactt ataatacagt tttttagttt
     3001 tgctggccgc atcttctcaa atatgcttcc cagcctgctt ttctgtaacg ttcaccctct
     3061 accttagcat cccttccctt tgcaaatagt cctcttccaa caataataat gtcagatcct
     3121 gtagagacca catcatccac ggttctatac tgttgaccca atgcgtctcc cttgtcatct
     3181 aaacccacac cgggtgtcat aatcaaccaa tcgtaacctt catctcttcc acccatgtct
     3241 ctttgagcaa taaagccgat aacaaaatct ttgtcgctct tcgcaatgtc aacagtaccc
     3301 ttagtatatt ctccagtaga tagggagccc ttgcatgaca attctgctaa catcaaaagg
     3361 cctctaggtt cctttgttac ttcttctgcc gcctgcttca aaccgctaac aatacctggg
     3421 cccaccacac cgtgtgcatt cgtaatgtct gcccattctg ctattctgta tacacccgca
     3481 gagtactgca atttgactgt attaccaatg tcagcaaatt ttctgtcttc gaagagtaaa
     3541 aaattgtact tggcggataa tgcctttagc ggcttaactg tgccctccat ggaaaaatca
     3601 gtcaagatat ccacatgtgt ttttagtaaa caaattttgg gacctaatgc ttcaactaac
     3661 tccagtaatt ccttggtggt acgaacatcc aatgaagcac acaagtttgt ttgcttttcg
     3721 tgcatgatat taaatagctt ggcagcaaca ggactaggat gagtagcagc acgttcctta
     3781 tatgtagctt tcgacatgat ttatcttcgt ttcctgcagg tttttgttct gtgcagttgg
     3841 gttaagaata ctgggcaatt tcatgtttct tcaacactac atatgcgtat atataccaat
     3901 ctaagtctgt gctccttcct tcgttcttcc ttctgttcgg agattaccga atcaaaaaaa
     3961 tttcaaggaa accgaaatca aaaaaaagaa taaaaaaaaa atgatgaatt gaaaaggtgg
     4021 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     4081 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     4141 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     4201 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     4261 tggtttctta gacggatcgc ttgcctgtaa cttacacgcg cctcgtatct tttaatgatg
     4321 gaataatttg ggaatttact ctgtgtttat ttatttttat gttttgtatt tggattttag
     4381 aaagtaaata aagaaggtag aagagttacg gaatgaagaa aaaaaaataa acaaaggttt
     4441 aaaaaatttc aacaaaaagc gtactttaca tatatattta ttagacaaga aaagcagatt
     4501 aaatagatat acattcgatt aacgataagt aaaatgtaaa atcacaggat tttcgtgtgt
     4561 ggtcttctac acagacaaga tgaaacaatt cggcattaat acctgagagc aggaagagca
     4621 agataaaagg tagtatttgt tggcgatccc cctagagtct tttacatctt cggaaaacaa
     4681 aaactatttt ttctttaatt tcttttttta ctttctattt ttaatttata tatttatatt
     4741 aaaaaattta aattataatt atttttatag cacgtgatga aaaggaccca ggtggcactt
     4801 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4861 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4921 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4981 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5041 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5101 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5161 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5221 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5281 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5341 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5401 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5461 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5521 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5581 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     5641 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5701 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5761 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5821 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5881 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5941 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6001 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6061 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6121 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6181 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6241 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6301 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6361 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6421 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6481 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6541 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     6601 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     6661 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     6721 agcgcccaat acgcaaac
//
