LOCUS       sequence-424940-        6491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative control plasmid for splicing reporters consisting of MCP 
            fused FLAG epitope tags.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6491)
  AUTHORS   Schmok JC, Jain M, Street LA, Tankka AT, Schafer D, Her HL, 
            Elmsaouri S, Gosztyla ML, Boyle EA, Jagannatha P, Luo EC, Kwon EJ, 
            Jovanovic M, Yeo GW
  TITLE     Large-scale evaluation of the ability of RNA-binding proteins to 
            activate exon inclusion.
  JOURNAL   Nat Biotechnol. 2024 Jan 2. doi: 10.1038/s41587-023-02014-0.
  PUBMED    38168984
REFERENCE   2  (bases 1 to 6491)
  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: "Nat 
            Biotechnol. 2024 Jan 2. doi: 10.1038/s41587-023-02014-0."
COMMENT     Sequence Label: FLAG NC
FEATURES             Location/Qualifiers
     source          1..6491
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1040..1161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1051..1070)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1105..1124
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1291..2313)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(1291..2313)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     protein_bind    complement(2321..2368)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     2500..2519
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2652..2876
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2865..2882
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3337..3378)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    3503..3527
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(3541..3564)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(3577..3600)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(3613..3636)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    complement(3658..3682)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3760..3779)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3761..3779)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3796..4974)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3805..4743
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3828..3848)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     5262..5281
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5356..5460
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5461..5529,5530..6321)
                     /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(5679..5698)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1081 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1141 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1201 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1261 ggcggtggaa tcgaaatctc gtagtacgtg ctattccttt gccctcggac gagtgctggg
     1321 gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct
     1381 tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca
     1441 tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg
     1501 gtcaagacca atgcggagca tatacgcccg gagccgcggc gatcctgcaa gctccggatg
     1561 cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa
     1621 gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc
     1681 tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg
     1741 ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga
     1801 cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc
     1861 gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa
     1921 cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atggcctccg cgaccggctg
     1981 cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg
     2041 ggagatgcaa taggtcaggc tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg
     2101 gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca
     2161 gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc
     2221 ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt
     2281 ctcgacagac gtcgcggtga gttcaggctt tttggccaag gaagttccta tactttctag
     2341 agaataggaa cttcggaata ggaacttcta ggtacgtgaa ccatcaccct aatcaagttt
     2401 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     2461 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     2521 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     2581 gcttaatgcg ccgctacagg gcgcgtgggg atacccccta gagccccagc tggttctttc
     2641 cgcctcagaa gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttcccaatc
     2701 ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca
     2761 atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt
     2821 caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga
     2881 ggctgatcag cgggtttaaa ctcattacta acccgtacga gatccacctc cacctccgta
     2941 gatgccggag ttggccgcga tggccgaggg aatcgggttt ccatctttta ggagaccttg
     3001 cattgcctta acaataagct cgcagtcgga attcgtggcg aaaattggaa tggttagttc
     3061 catatttaag taagatctcc atccggctac aggaagctct tcaccaccaa cagtctgggt
     3121 tgccacttta ggcacctcga ctttgatggt gtatttgcga ttctgcgcag agctctgacg
     3181 aacgctacag gttactttgt aagcctgtga tcgcgagtta gagctgatcc attcagcgac
     3241 cccgttagcg aagttgcttg gggcgacagt cacgtcgcca gttccgccat tgtcgacgag
     3301 aacgaactga gtaaagttag aagccatacc ggtacgcgta gaatcgagac cgaggagagg
     3361 gttagggata ggcttaccgg tagcgcttcc gccgcccccg ctgccgcctc cgccgctgcc
     3421 tcctcccccg cttccgccgc ctccgctggt ttcgaaccgc gggccctcta gactcgagcg
     3481 gccgccactg tgctggatat caaccacttt gtacaagaaa gctgggtaac taccacttcc
     3541 tttatcatcg tcatctttgt agtcgctgcc gctacctttg tcatcgtcgt ctttataatc
     3601 tgagccactt cccttatcgt catcgtcctt gtaatcagat ccacttccca tgctgccagc
     3661 ctgctttttt gtacaaactt gttgatatct gcagaattcc accacactgg actagtggat
     3721 ccgagctcgg taccaagctt acctagccag cttgggtctc cctatagtga gtcgtattag
     3781 taccaagcta attcctcacg acacctgaaa tggaagaaaa aaactttgaa ccactgtctg
     3841 aggcttgaga atgaaccaag atccaaactc aaaaagggca aattccaagg agaattacat
     3901 caagtgccaa gctggcctaa cttcagtctc cacccactca gtgtggggaa actccatcgc
     3961 ataaaacccc tccccccaac ctaaagacga cgtactccaa aagctcgaga actaatcgag
     4021 gtgcctggac ggcgcccggt actccgtgga gtcacatgaa gcgacggctg aggacggaaa
     4081 ggcccttttc ctttgtgtgg gtgactcacc cgcccgctct cccgagcgcc gcgtcctcca
     4141 ttttgagctc cctgcagcag ggccgggaag cggccatctt tccgctcacg caactggtgc
     4201 cgaccgggcc agccttgccg cccagggcgg ggcgatacac ggcggcgcga ggccaggcac
     4261 cagagcaggc cggccagctt gagactaccc ccgtccgatt ctcggtggcc gcgctcgcag
     4321 gccccgcctc gccgaacatg tgcgctggga cgcacgggcc ccgtcgccgc ccgcggcccc
     4381 aaaaaccgaa ataccagtgt gcagatcttg gcccgcattt acaagactat cttgccagaa
     4441 aaaaagcgtc gcagcaggtc atcaaaaatt ttaaatggct agagacttat cgaaagcagc
     4501 gagacaggcg cgaaggtgcc accagattcg cacgcggcgg ccccagcgcc caggccaggc
     4561 ctcaactcaa gcacgaggcg aaggggctcc ttaagcgcaa ggcctcgaac tctcccaccc
     4621 acttccaacc cgaagctcgg gatcaagaat cacgtactgc agccaggtgg aagtaattca
     4681 aggcacgcaa gggccataac ccgtaaagag gccaggcccg cgggaaccac acacggcact
     4741 tacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga ctactgcact
     4801 tatatacggt tctcccccac cctcgggaaa aaggcggagc cagtacacga catcactttc
     4861 ccagtttacc ccgcgccacc ttctctaggc accggttcaa ttgccgaccc ctccccccaa
     4921 cttctcgggg actgtgggcg atgtgcgctc tgcccactga cgggcaccgg agccaattcc
     4981 cactcctttc aagacctaga aggtccatta gctgcaaaga ttcctctctg tttaaaactt
     5041 tatccatctt tgcaaagctt tttgcaaaag cctagtcaat aatcaatgtc aacgcgtata
     5101 tctggcccgt acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat tcttcatgca
     5161 attgtcggtc aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact actcagcgac
     5221 ctccaacaca caagcaggga gcagatactg gcttaactat gcggcatcag agcagattgt
     5281 actgagagtg caccataggg gatcgggaga tctcccgatc cgtcgacgtc aggtggcact
     5341 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5401 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5461 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5521 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5581 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5641 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5701 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5761 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5821 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5881 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5941 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6001 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6061 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6121 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6181 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6241 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6301 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6361 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6421 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6481 aaaggatctt c
//
