LOCUS       sequence-367436-        7311 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RFA2-GFP fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7311)
  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 7311)
  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: KBB284
FEATURES             Location/Qualifiers
     source          1..7311
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     116..135
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     369..386
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    503..524
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(539..544,545..562,563..569)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    577..593
                     /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     582..604
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     601..617
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     601..617
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        638..656
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     684..700
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     685..701
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2183..2893
                     /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(2210..2238)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2833..2853
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2944..2962)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2958..3005
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3212..3231)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3213..3231)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3238..3255)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3238..3254)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3247..3269)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3395..3850
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3482..3501)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3692..3713
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3981..4784)
                     /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(4785..5000)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5024..5043)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5143..5165
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5203..5221)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    5262..5765
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        5802..5906
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5907..5975,5976..6767)
                     /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(6125..6144)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6938..7311,1..215)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
       61 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
      121 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
      181 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
      241 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
      301 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
      361 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
      421 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
      481 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
      541 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
      601 caggaaacag ctatgaccat gattacgcca agctcgaaat taaccctcac taaagggaac
      661 aaaagctggt accgggcccc ccctcgaggt cgacggtatc gataagcttg atatcgaatt
      721 cctgcaggcc cattttgaaa acctcaacgt taagaaagtc atttagagca gcaataacac
      781 tagatgtgga tatttctccc gaaatactaa tttttccctt aaatttgtcc acaaattgct
      841 gtactagctg gtatgattca ccactgatga tatgttcagc ctttttcaca tatgtctggt
      901 cgaaagaacc acttttcaga gattctactt ctgcagacag caataatttt gcatgcaata
      961 acgtctccat aattttgcaa ccactagttt actttttgcc tggtctgata gcttcttttt
     1021 agcaattcct ttggcctcga tgagcttcca ttttcaattt tcatcttttt tttcacgatg
     1081 cgaagtttct tagataatta tcgccatata cgaaacgcgt taggaaacgc gttctttcct
     1141 ttataataag caatattata tcaacttaag gtggaagtaa tgtggcaaaa tcgatagcga
     1201 ctatctagaa caggctagtt taagcatata cataatggca agtatgtatt agtgctagga
     1261 attggaggat ttgctctttt ctgcatcatt ttttttattt ttattggcga atgttaaaat
     1321 tactaacaat ggatgggatt ccaatacagc ctatcaacca tataacgaat attcatcagt
     1381 aacgggcggt ggctttgaga actctgagtc ccgcccaggt agtggggagt cggaaactaa
     1441 cactagagtt aacaccttga cacctgtgac gatcaaacaa attctagagt ccaaacagga
     1501 tattcaggac ggccccttcg tttcgcataa ccaagaactt catcacgttt gttttgtagg
     1561 tgtggtgaga aacattacag accatactgc aaatattttt ttaactattg aggatggaac
     1621 tggtcaaata gaagtgagaa aatggagcga agatgcaaat gacttggctg ccggtaacga
     1681 tgactcttct ggtaaaggtt atggttcgca agtcgcccaa caatttgaaa ttggcggtta
     1741 cgtaaaagtt tttggtgctt tgaaagagtt tggtggtaag aaaaatatac agtatgcggt
     1801 gattaagccc atagattcat tcaatgaagt gttgacgcat cacttggaag tcatcaaatg
     1861 tcattccata gccagtggaa tgatgaaaca acctttggag agtgcatcca acaacaatgg
     1921 gcaatcatta tttgtcaagg atgataacga tacatcttcc ggctccagtc cgttacaaag
     1981 aattctagaa ttttgtaaga agcaatgtga gggcaaagac gctaattcat tcgctgttcc
     2041 cattccattg atctcgcaat ccttgaattt ggatgaaact accgtcagaa actgctgtac
     2101 gaccttgact gaccagggtt ttatctaccc aacttttgat gacaataact tctttgccct
     2161 agctgctgga tccgctgctg ctagcaaagg agaagaactc ttcactggag ttgtcccaat
     2221 tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg gagagggtga
     2281 aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg gaaaactacc
     2341 tgttccatgg ccaacacttg tcactacttt cacttatggt gttcagtgct tttcaagata
     2401 cccggatcat atgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca
     2461 ggaaagaact atatttttca aagatgacgg gaactacaag acacgtgctg aagtcaagtt
     2521 tgaaggtgat acccttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg
     2581 aaacattctt gggcacaaat tggaatacaa ctataactca cacaatgtat acatcatggc
     2641 agacaaacaa aagaatggaa tcaaagctaa cttcaaaatt agacacaaca ttgaagatgg
     2701 aagcgttcaa ctagcagacc attatcaaca aaatactcca attggcgatg gccctgtcct
     2761 tttaccagac aaccattacc tgtccacaca atctgccctt tcgaaagatc ccaacgaaaa
     2821 gagagaccac atggtccttc ttgagtttgt aacagctgct gggattacac atggcatgga
     2881 tgaactatac aaaggatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     2941 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     3001 ttttgctgaa aggaggaact atatccggat atccacagga cgggtgtggt cgccatgatc
     3061 gcgtagtcga tagtggctcc aagtagcgaa gcgagcagga ctgggcggcg gccaaagcgg
     3121 tcggacagtg ctccgagaac gggtgcgcat agaaattgca tcaacgcata tagcgctaga
     3181 gcggccgcca ccgcggtgga gctccaattc gccctatagt gagtcgtatt acaattcact
     3241 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     3301 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     3361 ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc
     3421 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3481 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3541 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3601 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3661 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3721 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3781 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3841 cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3901 tagggtaata actgatataa ttaaattgaa gctctaattt gtgagtttag tatacatgca
     3961 tttacttata atacagtttt ttagttttgc tggccgcatc ttctcaaata tgcttcccag
     4021 cctgcttttc tgtaacgttc accctctacc ttagcatccc ttccctttgc aaatagtcct
     4081 cttccaacaa taataatgtc agatcctgta gagaccacat catccacggt tctatactgt
     4141 tgacccaatg cgtctccctt gtcatctaaa cccacaccgg gtgtcataat caaccaatcg
     4201 taaccttcat ctcttccacc catgtctctt tgagcaataa agccgataac aaaatctttg
     4261 tcgctcttcg caatgtcaac agtaccctta gtatattctc cagtagatag ggagcccttg
     4321 catgacaatt ctgctaacat caaaaggcct ctaggttcct ttgttacttc ttctgccgcc
     4381 tgcttcaaac cgctaacaat acctgggccc accacaccgt gtgcattcgt aatgtctgcc
     4441 cattctgcta ttctgtatac acccgcagag tactgcaatt tgactgtatt accaatgtca
     4501 gcaaattttc tgtcttcgaa gagtaaaaaa ttgtacttgg cggataatgc ctttagcggc
     4561 ttaactgtgc cctccatgga aaaatcagtc aagatatcca catgtgtttt tagtaaacaa
     4621 attttgggac ctaatgcttc aactaactcc agtaattcct tggtggtacg aacatccaat
     4681 gaagcacaca agtttgtttg cttttcgtgc atgatattaa atagcttggc agcaacagga
     4741 ctaggatgag tagcagcacg ttccttatat gtagctttcg acatgattta tcttcgtttc
     4801 ctgcaggttt ttgttctgtg cagttgggtt aagaatactg ggcaatttca tgtttcttca
     4861 acactacata tgcgtatata taccaatcta agtctgtgct ccttccttcg ttcttccttc
     4921 tgttcggaga ttaccgaatc aaaaaaattt caaggaaacc gaaatcaaaa aaaagaataa
     4981 aaaaaaaatg atgaattgaa aaggtggtat ggtgcactct cagtacaatc tgctctgatg
     5041 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     5101 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     5161 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     5221 ttttataggt taatgtcatg ataataatgg tttcttagac ggatcgcttg cctgtaactt
     5281 acacgcgcct cgtatctttt aatgatggaa taatttggga atttactctg tgtttattta
     5341 tttttatgtt ttgtatttgg attttagaaa gtaaataaag aaggtagaag agttacggaa
     5401 tgaagaaaaa aaaataaaca aaggtttaaa aaatttcaac aaaaagcgta ctttacatat
     5461 atatttatta gacaagaaaa gcagattaaa tagatataca ttcgattaac gataagtaaa
     5521 atgtaaaatc acaggatttt cgtgtgtggt cttctacaca gacaagatga aacaattcgg
     5581 cattaatacc tgagagcagg aagagcaaga taaaaggtag tatttgttgg cgatccccct
     5641 agagtctttt acatcttcgg aaaacaaaaa ctattttttc tttaatttct ttttttactt
     5701 tctattttta atttatatat ttatattaaa aaatttaaat tataattatt tttatagcac
     5761 gtgatgaaaa ggacccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5821 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5881 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5941 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     6001 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     6061 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     6121 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     6181 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     6241 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6301 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6361 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6421 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6481 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6541 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6601 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6661 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6721 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6781 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6841 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6901 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6961 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     7021 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     7081 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     7141 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     7201 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     7261 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg a
//
