LOCUS       sequence-408143-        8997 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mEGFP-tagged fusion protein, MKRN1_BRAF from patient-derived
            sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8997)
  AUTHORS   Tripathi S, Shirnekhi HK, Gorman SD, Chandra B, Baggett DW, Park CG,
            Somjee R, Lang B, Hosseini SMH, Pioso BJ, Li Y, Iacobucci I, Gao Q, 
            Edmonson MN, Rice SV, Zhou X, Bollinger J, Mitrea DM, White MR, 
            McGrail DJ, Jarosz DF, Yi SS, Babu MM, Mullighan CG, Zhang J, Sahni 
            N, Kriwacki RW
  TITLE     Defining the condensate landscape of fusion oncoproteins.
  JOURNAL   Nat Commun. 2023 Sep 28;14(1):6008. doi: 10.1038/s41467-023-41655-2.
  PUBMED    37770423
REFERENCE   2  (bases 1 to 8997)
  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 
            Commun."; date: "2023-09-28"; volume: "14(1):6008. doi"; pages: " 
            10.1038/s41467-023-41655-2"
COMMENT     Sequence Label: CL20_mEGFP_MKRN1_BRAF
FEATURES             Location/Qualifiers
     source          1..8997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     220..239
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     473..490
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    558..692
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(582..601)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     636..655
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(698..893)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      712..847
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    952..973
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(988..993,994..1011,1012..1018)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1026..1042
                     /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     1031..1053
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1050..1066
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1050..1066
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1409..1428
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    1427..1482
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     1528..1547
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(join(3758..4468,4469..4471,4472..4474))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3800..3821)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4149..4168
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4408..4429
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      4471..4480
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4556..4759)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4585..4605)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4760..5063
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     CDS             complement(5320..5364)
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    5549..5782
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    6142..6259
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_feature    6751..6876
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             6923..7180
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     enhancer        7182..7561
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7592..7696
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7697..7765,7766..8557)
                     /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(7915..7934)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(8728..8997,1..319)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
       61 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
      121 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
      181 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
      241 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
      301 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
      361 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
      421 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
      481 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
      541 gattcattaa tgcagcggat ccagacatga taagatacat tgatgagttt ggacaaacca
      601 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
      661 ttgtaaccat tataagctgc aataaacaag ttagcttttt gcaaaagcct aggcctccaa
      721 aaaagcctcc tcactacttc tggaatagct cagaggccga ggcggcctcg gcctctgcat
      781 aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg
      841 gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga tgcggatccg
      901 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
      961 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     1021 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     1081 gcttgggctg caggtcgagg gatctccata agagaagagg gacagctatg actgggagta
     1141 gtcaggagag gaggaaaaat ctggctagta aaacatgtaa ggaaaatttt agggatgtta
     1201 aagaaaaaaa taacacaaaa caaaatataa aaaaaatcta acctcaagtc aaggcttttc
     1261 tatggaataa ggaatggaca gcagggggct gtttcatata ctgatgacct ctttatagcc
     1321 aacctttgtt catggcagcc agcatatggg catatgttgc caaactctaa accaaatact
     1381 cattctgatg ttttaaatga tttgccctcc catatgtcct tccgagtgag agacacaaaa
     1441 aattccaaca cactattgca atgaaaataa atttccttta ttagccagaa gtcagatgct
     1501 caaggggctt catgatgtcc ccataatttt tggcagaggg aaaaagatct gctagctcga
     1561 gcagctgaag ctttattgag gcttaagcag tgggttccct agttagccag agagctccca
     1621 ggctcagatc tggtctaacc agagagaccc agtacaggca aaaagcagat cttgtcttct
     1681 ttgggagtga attagccctt ccagtccccc cttttctttt aaaaagtggc taagatctac
     1741 agctgccttg taagtcattg gtcttaaagg taccgtaatc ttatctagac taatggactg
     1801 ggaaggctcc atatccacct gcctggatcg gggttttcgg agatgcacac gcataaagac
     1861 tgaaatcttc cgtttgaaaa cctgcgcggt ttaaagaagg ttcggaggcc gagcgatgaa
     1921 tctttggcaa cgagcgcgcc agcaattcaa tagatgccag aatttggggg aaaagtggac
     1981 gctcatcacg tttttttttc aagcattcag ccataagacg tttcatggct ttcgggcagt
     2041 tcgaacggac cttggaaaga tccggactca gatagccgcg cccaaccatg aagataattt
     2101 gatcgcggtt attgatattt gaatacggta actggcctgt cataagttca tacaatacga
     2161 tgccgaatgc gtagacgtct gactgaaaag agtaagggtt tttgtcctgc atgcgaatta
     2221 cctccggagc catccaaagg attgagccag aaagttgctc gaattgatgc gatccagacc
     2281 agcggctctt caccgtcgca agcccaaaat ccccaatttt taccgtaaga tcctcatgta
     2341 agaagatgtt gttggacttt aaatcgcggt gaatgatcga ttttgcatgc agatagtcca
     2401 tgccttgcgc agtttgacgt gcaatgtcga taagtttaat catttcaaat ttagtttcaa
     2461 taatatgtaa atggtggtac agagacgaac cctcacacca ttgagtaacg attgccaatt
     2521 gcggcttcgt actatatccc ataaacagaa gaatatttac gtgacgtgtc ttgcgcagga
     2581 ccccgacctc gtttttgaac gcttgcaatt gctgtggagt cggcgcggtt acgttcagca
     2641 tttttacggc cacatcaccg tgccatttgc ccttgtacac cgtgccaaaa gaaccggagc
     2701 caatgcgctg acccactgta atttggccgt cggggatctc ccagtcgtca gaactatcgc
     2761 ggcgaccaag ggtcttcata cgattacgat cctcggacga gctgctggac ttgcgctcac
     2821 gttgcggtcc cggcgacttt tgaagggctt tcacattggt cagcgatcca ggtaaactag
     2881 cggggggtgt tgcggacagt cctgtggtcg aaccgccatc tccgcgaaag ccctgatcac
     2941 ggattaagtc cttgatgtgt tgagaacgct gcgcagcatc cattggatgt aagacctgta
     3001 aaccacacat atcacaggag tcgccgtgta agtagacgca attctcaccg tagcggcatt
     3061 ctcctactgc cgcataggga caaagttgtt tctttgtctc aacggcagtt tgctctttct
     3121 ccgactcctc cttagtaaca ctgccttgta aaggcgcttc ggtacaggaa ggagcagtgc
     3181 gtccacagta tggttgaccg gggacaaact cgatggcatt gacccaatcc tcgctgccag
     3241 ccccgacagt ggcgaaattc gaattacgac tttcggcctc tcccgtattc atctcgacca
     3301 aaggacctac aatactcgaa agagagctag aggctgccag actagattta gtagtcagct
     3361 ctgtggctgt agcctcttct tgttttaatg gtttcgagtg ttcataacga cagcggtcgc
     3421 cataaatgca gtaaccgcgt tgaaagtact tgcacactac actgtatgga gagtcactta
     3481 aatcgtggga ataacgacaa ttgtccccct ccttgcagac cccatgcatg aagtagcgac
     3541 acgtcacctg tttagtccag cctccccctg agccgtctga accgccaccc ccgccaccag
     3601 ctccaagact cggagcggtt actgtcggga tcggtgtcgg gcttgctgcg gcagcggtcg
     3661 ctgcggctgc tccagctcct gacgttgtcg cagttgttcc aggagtcgct gcctccgcca
     3721 tagcggccgc acttcccccg gagcctccgc ttccgccctt gtacagctcg tccatgccga
     3781 gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg
     3841 ggtctttgct cagcttggac tgggtgctca ggtagtggtt gtcgggcagc agcacggggc
     3901 cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga
     3961 tgttgtggcg gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg gccatgatat
     4021 agacgttgtg gctgttgtag ttgtactcca gcttgtgccc caggatgttg ccgtcctcct
     4081 tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct
     4141 cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc
     4201 cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcggctga
     4261 agcactgcac gccgtaggtc agggtggtca cgagggtggg ccagggcacg ggcagcttgc
     4321 cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc
     4381 cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca
     4441 ccccggtgaa cagctcctcg cccttgctca ccatggtggc gaccggtgga tcccgggccc
     4501 gcggtaccgt cgactgcaga attcggttca ctaaacgagc ccggttcact aaacgagctc
     4561 tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt
     4621 tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt
     4681 caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt
     4741 actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag
     4801 taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat
     4861 tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag
     4921 tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg
     4981 gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc
     5041 catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg
     5101 taattgatta ctattaataa ctagcacgcg ttcgaagctt gagctcggtc ccctcggggt
     5161 tgggaggtgg gtctgaaacg ataatggtga atatccctgc ctaactctat tcactataga
     5221 aagtacagca aaaactattc ttaaacctac caagcctcct actatcatta tgaataattt
     5281 tatataccac agccaatttg ttatgttaaa ccaattccac aaacttgccc atttatctaa
     5341 ttccaataat tcttgttcat tcttttcttg ctggttttgc gattcttcaa ttaaggagtg
     5401 tattaagctt gtgtaattgt taatttctct gtcccactcc atccaggtcg tgtgattcca
     5461 aatctgttcc agagatttat tactccaact agcattccaa ggcacagcag tggtgcaaat
     5521 gagttttcca gagcaacccc aaatccccag gagctgttga tcctttaggt atctttccac
     5581 agccaggatt cttgcctgga gctgcttgat gccccagact gtgagttgca acagatgctg
     5641 ttgcgcctca atagccctca gcaaattgtt ctgctgctgc actataccag acaataattg
     5701 tctggcctgt accgtcagcg tcattgacgc tgcgcccata gtgcttcctg ctgctcccaa
     5761 gaacccaagg aacaaagctc ctattcccac tgctcttttt tctctctgca ccactcttct
     5821 ctttgccttg gtgggtgcta ctcctaatgg ttcaattttt actactttat atttatataa
     5881 ttcacttctc caattgtccc tcatatctcc tccttatggt gttttactaa acttttccat
     5941 gttctaatcc tcatcctgtc tacttgccac acaatcatca cctgccatct gttttccata
     6001 atccctaatg atctttgctt ttcttcttgg cactactttt atgtcactat tatcttgtat
     6061 tactactgcc ccttcacctt tccagaggag ctttgctggt cctttccaaa gtggatttct
     6121 gctgtccctg taataaaccc gaaaattttg aatttttgta atttgttttt gtaattcttt
     6181 agtttgtatg tctgttgcta ttatgtctac tattctttcc cctgcactgt accccccaat
     6241 cccccctttt cttttaaaat tgtggatgaa tactgccatt tgtactgctg tcttaagatg
     6301 ttcagcctga tctcttacct gtcctataat tttctttaat tctttattca tagattctac
     6361 tactccttga ctttggggat tgtagggaat ttccaggtct gaagatcctg ctgcttgctg
     6421 tgcttttttc ttacttttgt tttgctcttc ctctatcttg tctaaagctt ccttggtgtc
     6481 ttttatctct atcctttgat gcacacaata gagggttgct actgtattat ataatgatct
     6541 aagttcttct gatcctgtct gaagggatgg ttgtagctgt cccagtattt gtctacagcc
     6601 ttctgatgtt tctaacaggc cagtattaac tgcgaatcgt tctagctccc tgcttgccca
     6661 tactatatgt tttaatttat attttttctt tccccctggc cttaaccgaa ttttttccca
     6721 tcgcgatcta attctccccc gcttaatact gacgctctcg cacccatctc tctccttcta
     6781 gcctccgcta gtcaaaattt ttggcgtact caccagtcgc cgcccctcgc ctcttgccgt
     6841 gcgcgcttca gcaagccgag tcctgcgtcg agagagctcc tctggtttcc ctttcgcttt
     6901 caagtccctg ttcgggcgcc actgctagag attttccaca ctgactaaaa gggtctgagg
     6961 gatctctagt taccagagtc acacaacaga cgggcacaca ctacttgaag cactcaaggc
     7021 aagctttatt gaggcttaag cagtgggttc cctagttagc cagagagctc ccaggctcag
     7081 atctggtcta accagagaga cccagtacag gcaaaaagca gctgcttata tgcaggatct
     7141 gagggctcgc cactccccag tcccgcccag gccacgcctc ccatggtaat agcgatgact
     7201 aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc
     7261 caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact
     7321 tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa
     7381 gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg
     7441 ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat
     7501 gggctatgaa ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt
     7561 cgacccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     7621 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     7681 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     7741 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     7801 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     7861 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     7921 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     7981 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     8041 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     8101 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     8161 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     8221 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     8281 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     8341 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     8401 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     8461 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     8521 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     8581 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     8641 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     8701 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     8761 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     8821 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     8881 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     8941 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgg
//
