LOCUS       sequence-150806-        6853 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Derived from FRE5. A two nucleotide insertion to shift the reading 
            frame to dsRED..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6853)
  AUTHORS   Orengo JP, Bundman D, Cooper TA
  TITLE     A bichromatic fluorescent reporter for cell-based screens of 
            alternative splicing.
  JOURNAL   Nucleic Acids Res. 2006;34(22):e148. Epub 2006 Nov 16.
  PUBMED    17142220
REFERENCE   2  (bases 1 to 6853)
  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"; journalName: "Nucleic 
            Acids Res."; date: "2006"; volume: "34(22)"; pages: "e148. Epub 2006
            Nov 16"
COMMENT     Sequence Label: FRE5Cf
FEATURES             Location/Qualifiers
     source          1..6853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             922..945
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             958..978
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1015..1017,1018..1692)
                     /codon_start=1
                     /product="wild-type DsRed"
                     /label=DsRed1
                     /note="mammalian codon-optimized"
                     /translation="VRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(1195..1215)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1413..1432
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1607..1630
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             join(1701..1703,1704..2417)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1701..1703,1704..2414)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1743..1764)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2004..2023)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2351..2372
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2443..2460)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2449..2673
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2719..3147
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2806..2825)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3016..3037
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3156..3176)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3161..3491
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3342..3477
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3404..3423
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3558..4352
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3612..3631)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4222..4241
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4526..4647
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4563..4582)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4617..4636
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4696..4712)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4696..4712)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4709..4731)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4720..4736
                     /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)."
     promoter        complement(join(4744..4750,4751..4768,4769..4774))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4789..4810
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4927..4944)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5098..5686)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5178..5197)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5857..6648,6649..6717))
                     /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     6480..6499
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6718..6822)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttccat ggattacaag gatgacgatg acaagggggt acctgcccca
      961 aaaaaaaaac gcaaagtgga ggacccagta ccaggatcca atcgcgatac cggtgtgcgc
     1021 tcctccaaga acgtcatcaa ggagttcatg cgcttcaagg tgcgcatgga gggcaccgtg
     1081 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggccacaac
     1141 accgtgaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc
     1201 ccccagttcc agtacggctc caaggtgtac gtgaagcacc ccgccgacat ccccgactac
     1261 aagaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     1321 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gctgcttcat ctacaaggtg
     1381 aagttcatcg gcgtgaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     1441 tgggaggcct ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg cgagatccac
     1501 aaggccctga agctgaagga cggcggccac tacctggtgg agttcaagtc catctacatg
     1561 gccaagaagc ccgtgcagct gcccggctac tactacgtgg actccaagct ggacatcacc
     1621 tcccacaacg aggactacac catcgtggag cagtacgagc gcaccgaggg ccgccaccac
     1681 ctgttcctgt agaccgcggt gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1741 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1801 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1861 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     1921 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1981 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     2041 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     2101 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     2161 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     2221 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     2281 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     2341 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     2401 acgagctgta caagtaaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     2461 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2521 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     2581 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     2641 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     2701 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2761 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2821 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cggggcatcc
     2881 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2941 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3001 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3061 tctattcttt tgatttataa gggattttgg ggatttcggc ctattggtta aaaaatgagc
     3121 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     3181 aaagtcccca ggctccccag gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3241 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3301 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3361 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3421 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3481 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     3541 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3601 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     3661 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     3721 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     3781 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     3841 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     3901 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     3961 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4021 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4081 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4141 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4201 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4261 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4321 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     4381 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     4441 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4501 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     4561 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4621 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     4681 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4741 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4801 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4861 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4921 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4981 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5041 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5101 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5161 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5221 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5281 ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5341 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5401 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5461 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5521 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5581 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5641 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5701 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5761 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5821 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5881 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5941 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6001 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6061 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6121 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6181 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6241 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6301 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6361 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6421 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6481 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6541 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6601 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6661 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6721 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6781 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6841 gccacctgac gtc
//
