LOCUS       sequence-150805-        6851 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter to quantify the ratio of two alternative 
            splicing patterns..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6851)
  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 6851)
  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: FRE5
FEATURES             Location/Qualifiers
     source          1..6851
                     /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(1013..1015,1016..1690)
                     /codon_start=1
                     /product="wild-type DsRed"
                     /label=DsRed1
                     /note="mammalian codon-optimized"
                     /translation="VRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(1193..1213)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1411..1430
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1605..1628
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             join(1699..1701,1702..2415)
                     /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(1699..1701,1702..2412)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1741..1762)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2002..2021)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2349..2370
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2441..2458)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2447..2671
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2717..3145
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2804..2823)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3014..3035
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3154..3174)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3159..3489
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3340..3475
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3402..3421
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3556..4350
                     /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(3610..3629)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4220..4239
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4524..4645
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4561..4580)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4615..4634
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4694..4710)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4694..4710)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4707..4729)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4718..4734
                     /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(4742..4748,4749..4766,4767..4772))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4787..4808
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4925..4942)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5096..5684)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5176..5195)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5855..6646,6647..6715))
                     /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     6478..6497
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6716..6820)
                     /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 atcgataccg gtgtgcgctc
     1021 ctccaagaac gtcatcaagg agttcatgcg cttcaaggtg cgcatggagg gcaccgtgaa
     1081 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gccacaacac
     1141 cgtgaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca tcctgtcccc
     1201 ccagttccag tacggctcca aggtgtacgt gaagcacccc gccgacatcc ccgactacaa
     1261 gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg
     1321 cgtggtgacc gtgacccagg actcctccct gcaggacggc tgcttcatct acaaggtgaa
     1381 gttcatcggc gtgaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg
     1441 ggaggcctcc accgagcgcc tgtacccccg cgacggcgtg ctgaagggcg agatccacaa
     1501 ggccctgaag ctgaaggacg gcggccacta cctggtggag ttcaagtcca tctacatggc
     1561 caagaagccc gtgcagctgc ccggctacta ctacgtggac tccaagctgg acatcacctc
     1621 ccacaacgag gactacacca tcgtggagca gtacgagcgc accgagggcc gccaccacct
     1681 gttcctgtag accgcggtgt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     1741 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     1801 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1861 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     1921 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1981 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     2041 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     2101 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     2161 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     2221 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2281 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     2341 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     2401 gagctgtaca agtaagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     2461 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     2521 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     2581 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     2641 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     2701 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2761 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2821 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg gggcatccct
     2881 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2941 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3001 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     3061 tattcttttg atttataagg gattttgggg atttcggcct attggttaaa aaatgagctg
     3121 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     3181 agtccccagg ctccccaggc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3241 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3301 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3361 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3421 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3481 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     3541 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3601 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3661 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3721 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3781 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3841 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3901 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3961 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     4021 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     4081 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     4141 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     4201 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4261 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4321 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4381 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4441 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4501 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4561 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4621 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4681 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4741 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4801 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4861 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4921 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4981 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5041 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5101 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5161 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5221 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5281 cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5341 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5401 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5461 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5521 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5581 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5641 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5701 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5761 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5821 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5881 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5941 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6001 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6061 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6121 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6181 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6241 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6301 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6361 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6421 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6481 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6541 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6601 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6661 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6721 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6781 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6841 cacctgacgt c
//
