LOCUS       sequence-368137-        6244 bp    DNA     circular SYN 15-APR-2025
DEFINITION  rfa3D46 truncation fused to GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6244)
  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 6244)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: KBB345
FEATURES             Location/Qualifiers
     source          1..6244
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..804)
                     /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(805..1020)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(1044..1063)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1163..1185
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1223..1241)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    1282..1785
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        1822..1926
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1927..1995,1996..2787)
                     /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(2145..2164)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2958..3546
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3447..3466
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3700..3717
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3834..3855
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3870..3875,3876..3893,3894..3900)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3908..3924
                     /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     3913..3935
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3932..3948
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3932..3948
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3969..3987
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     4015..4031
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4016..4032
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4447..5157
                     /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(4474..4502)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5097..5117
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(5208..5226)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5222..5269
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(5476..5495)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5477..5495)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5502..5519)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5502..5518)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5511..5533)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5659..6114
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5746..5765)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5956..5977
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ttagttttgc tggccgcatc ttctcaaata tgcttcccag cctgcttttc tgtaacgttc
       61 accctctacc ttagcatccc ttccctttgc aaatagtcct cttccaacaa taataatgtc
      121 agatcctgta gagaccacat catccacggt tctatactgt tgacccaatg cgtctccctt
      181 gtcatctaaa cccacaccgg gtgtcataat caaccaatcg taaccttcat ctcttccacc
      241 catgtctctt tgagcaataa agccgataac aaaatctttg tcgctcttcg caatgtcaac
      301 agtaccctta gtatattctc cagtagatag ggagcccttg catgacaatt ctgctaacat
      361 caaaaggcct ctaggttcct ttgttacttc ttctgccgcc tgcttcaaac cgctaacaat
      421 acctgggccc accacaccgt gtgcattcgt aatgtctgcc cattctgcta ttctgtatac
      481 acccgcagag tactgcaatt tgactgtatt accaatgtca gcaaattttc tgtcttcgaa
      541 gagtaaaaaa ttgtacttgg cggataatgc ctttagcggc ttaactgtgc cctccatgga
      601 aaaatcagtc aagatatcca catgtgtttt tagtaaacaa attttgggac ctaatgcttc
      661 aactaactcc agtaattcct tggtggtacg aacatccaat gaagcacaca agtttgtttg
      721 cttttcgtgc atgatattaa atagcttggc agcaacagga ctaggatgag tagcagcacg
      781 ttccttatat gtagctttcg acatgattta tcttcgtttc ctgcaggttt ttgttctgtg
      841 cagttgggtt aagaatactg ggcaatttca tgtttcttca acactacata tgcgtatata
      901 taccaatcta agtctgtgct ccttccttcg ttcttccttc tgttcggaga ttaccgaatc
      961 aaaaaaattt caaggaaacc gaaatcaaaa aaaagaataa aaaaaaaatg atgaattgaa
     1021 aaggtggtat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     1081 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     1141 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     1201 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     1261 ataataatgg tttcttagac ggatcgcttg cctgtaactt acacgcgcct cgtatctttt
     1321 aatgatggaa taatttggga atttactctg tgtttattta tttttatgtt ttgtatttgg
     1381 attttagaaa gtaaataaag aaggtagaag agttacggaa tgaagaaaaa aaaataaaca
     1441 aaggtttaaa aaatttcaac aaaaagcgta ctttacatat atatttatta gacaagaaaa
     1501 gcagattaaa tagatataca ttcgattaac gataagtaaa atgtaaaatc acaggatttt
     1561 cgtgtgtggt cttctacaca gacaagatga aacaattcgg cattaatacc tgagagcagg
     1621 aagagcaaga taaaaggtag tatttgttgg cgatccccct agagtctttt acatcttcgg
     1681 aaaacaaaaa ctattttttc tttaatttct ttttttactt tctattttta atttatatat
     1741 ttatattaaa aaatttaaat tataattatt tttatagcac gtgatgaaaa ggacccaggt
     1801 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     1861 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     1921 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     1981 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2041 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2101 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     2161 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     2221 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     2281 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     2341 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     2401 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     2461 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     2521 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     2581 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     2641 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     2701 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     2761 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     2821 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     2881 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2941 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3001 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3061 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     3121 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3181 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3241 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3301 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3361 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3421 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3481 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3541 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3601 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3661 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3721 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     3781 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     3841 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     3901 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     3961 aagctcgaaa ttaaccctca ctaaagggaa caaaagctgg taccgggccc cccctcgagg
     4021 tcgacggtat cgataagctt gatatcgaat tcctgcaggc aacgtcaaga acagatgaca
     4081 gccaacagtc gttgactaac gtaatgggcc actgctgaga agcatccacc gcccatgcca
     4141 tggttggaat catgacaagt cgccctatat aatgccttcg ttctttaata ttgacgcgcc
     4201 aaatgtggca atacgcgata tttttttcgt tcgtgtttaa ataaagagca atataacaca
     4261 cccttcacca ttcttgtaga acatcgtcta ccagtaacac aagtaaaaat ggccagcgaa
     4321 acaccaagag ttgaccccac agaaatctcc aacgtcaatg ctcctgtgtt taggataata
     4381 gcgcaaatca aatcacaacc cactgaatct cagttgattt tacaatcgcc aaccatatca
     4441 tcgaaaagca aaggagaaga actcttcact ggagttgtcc caattcttgt tgaattagat
     4501 ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac
     4561 ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca
     4621 cttgtcacta ctttcactta tggtgttcag tgcttttcaa gatacccgga tcatatgaaa
     4681 cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaaag aactatattt
     4741 ttcaaagatg acgggaacta caagacacgt gctgaagtca agtttgaagg tgataccctt
     4801 gttaatagaa tcgagttaaa aggtattgat tttaaagaag atggaaacat tcttgggcac
     4861 aaattggaat acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat
     4921 ggaatcaaag ctaacttcaa aattagacac aacattgaag atggaagcgt tcaactagca
     4981 gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat
     5041 tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga ccacatggtc
     5101 cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact atacaaagga
     5161 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     5221 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     5281 aactatatcc ggatatccac aggacgggtg tggtcgccat gatcgcgtag tcgatagtgg
     5341 ctccaagtag cgaagcgagc aggactgggc ggcggccaaa gcggtcggac agtgctccga
     5401 gaacgggtgc gcatagaaat tgcatcaacg catatagcgc tagagcggcc gccaccgcgg
     5461 tggagctcca attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa
     5521 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     5581 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     5641 agcctgaatg gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     5701 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     5761 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5821 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     5881 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5941 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     6001 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     6061 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat
     6121 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatagggt aataactgat
     6181 ataattaaat tgaagctcta atttgtgagt ttagtataca tgcatttact tataatacag
     6241 tttt
//
