LOCUS       sequence-396310-        7538 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A fluorescent reporter for SF3B1 K700E mutation. Co-expresses a 
            blasticidin resistance gene for selection. Includes LoxP sites for 
            Cre-mediated deletion of reporter cassette. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7538)
  AUTHORS   Fernandez M, Yu L, Jia Q, Wang X, Hart KL, Jia Z, Lin RJ, Wang L
  TITLE     Engineering oncogenic hotspot mutations on SF3B1 via CRISPR-directed
            PRECIS mutagenesis.
  JOURNAL   Cancer Res Commun. 2024 Aug 28. doi: 10.1158/2767-9764.CRC-24-0145.
  PUBMED    39194178
REFERENCE   2  (bases 1 to 7538)
  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: "Cancer Res 
            Commun. 2024 Aug 28. doi: 10.1158/2767-9764.CRC-24-0145."
COMMENT     Sequence Label: pLenti-SF3B1-K700E-reporter
FEATURES             Location/Qualifiers
     source          1..7538
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        58..361
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        362..565
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     516..536
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             580..760
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    804..929
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1422..1655
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1840..1884
                     /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    2151..2268
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2383..2416)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2478..2499)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2827..2846
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3236..3257
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(3303..3359)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(3375..3770)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      3767..3776
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3797..4008)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    4022..4610
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4075..4095)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4493..4504)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(4627..4660)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     LTR             4813..5046
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     rep_origin      5307..5442
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     rep_origin      complement(5560..6148)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5640..5659)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(6510..6578,6579..7370)
                     /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(6728..6747)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      7383..7519
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
ORIGIN
        1 gcgatcgcag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
       61 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      121 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      181 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      241 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      301 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      361 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      421 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      481 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      541 gtgggaggtc tatataagca gagctcgttt agtgaaccgg ggtctctctg gttagaccag
      601 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
      661 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
      721 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggacc
      781 tgaaagcgaa agggaaacca gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      841 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      901 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      961 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
     1021 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
     1081 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
     1141 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
     1201 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
     1261 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     1321 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1381 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1441 ttcttgggag cagcaggaag cactatgggc gcagcctcaa tgacgctgac ggtacaggcc
     1501 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1561 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1621 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1681 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1741 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1801 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1861 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1921 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1981 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     2041 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     2101 agagacagat ccattcgatt agtgaacgga tctcgacggt atcggttaac ttttaaaaga
     2161 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
     2221 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttgg ctcccgatcg
     2281 ttgcgttaca cacacaatta ctgctgatcg agtgtagcct tcgaatgatc attgtcgatc
     2341 ctaccatcca ctcgacacac ccgccaggat ccttttgaat aaataacttc gtatagcata
     2401 cattatacga agttatctat ccgggtacta gtttacttgt acagctcgtc catgccgaga
     2461 gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg
     2521 tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag cacggggccg
     2581 tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg
     2641 ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc catgatatag
     2701 acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg
     2761 aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg
     2821 gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg caataaactt
     2881 gccaatggca ttttaggata aagacaaagt aaaataaaac atagaaatgc cttaccatgg
     2941 cctacaaggc ctctttgggc tctgccacca ccctaattcc catttccctc tcggagcaga
     3001 ggaagggggc gactcacctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc
     3061 tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg
     3121 agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg
     3181 ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg
     3241 ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc
     3301 atcggtccag gattctcttc gacatctccg gcttgtttca gcagagagaa gtttgttgcg
     3361 ccgctgccgc tagcgccctc ccacacataa ccagagggca gcaattcacg aatcccaact
     3421 gccgtcggct gtccatcact gtccttcact atggctttga tcccaggatg cagatcgaga
     3481 agcacctgtc ggcaccgtcc gcaggggctc aagatgcccc tgttctcatt tccgatcgcg
     3541 acgatacaag tcaggttgcc agctgccgca gcagcagcag tgcccagcac cacgagttct
     3601 gcacaaggtc ccccagtaaa atgatataca ttgacaccag tgaagatgcg gccgtcgcta
     3661 gagagagctg cgctggcgac gctgtagtct tcagagatgg ggatgctgtt gattgtagcc
     3721 gttgctcttt caatgagggt ggattcttct tgagacaaag gcttggccat ggtggctcta
     3781 gacttgggct gcaggtctgt gttctggcgg caaacccgtt gcgaaaaaga acgttcacgg
     3841 cgactactgc acttatatac ggttctcccc caccctcggg aaaaaggcgg agccagtaca
     3901 cgacatcact ttcccagttt accccgcgcc accttctcta ggcaccggtt caattgccga
     3961 cccctccccc caacttctcg gggactgtgg gcgatgtgcg ctctgcccac gcgtctggaa
     4021 caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     4081 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     4141 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     4201 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     4261 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     4321 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     4381 gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc catggctgct
     4441 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     4501 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     4561 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc cgtacgctaa
     4621 acgaaaataa cttcgtatag catacattat acgaagttat tcacttacag ctcgagggcc
     4681 cgcatccacc atcgcagact tatcatctta ttccagatgc gtgcggatgg aattcgagct
     4741 cggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     4801 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
     4861 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     4921 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     4981 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     5041 ctagcagtcc tggccaacgt gagcaccgtg ctgacctcca aatatcgtta agctggagcc
     5101 tgggagccgg cctggccctc cgcccccccc acccccgcag cccacccctg gtctttgaat
     5161 aaagtctgag tgagtggccg acagtgcccg tggagttctc gtgacctgag gtgcagggcc
     5221 ggcgctaggg acacgtccgt gcacgtgccg aggccccctg tgcagctgca agggacaggc
     5281 ctagccctgc aggcctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     5341 ctccgcctca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     5401 ctgagctatt ccagaagtag tgaggacgct tttttggagg ccgaggcttt tgcaaagatc
     5461 gaacaagaga caggacctgc aggttaatta aatttaaatc atgtgagcaa aaggccagca
     5521 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5581 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5641 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5701 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5761 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5821 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5881 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5941 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6001 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6061 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6121 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6181 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6241 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6301 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6361 tctatttcgt tcatccatag ttgcatttaa atggccggcc tggcgcgccg tttaaaccta
     6421 gatattgata gtctgatcgg tcaacgtata atcgagtcct agcttttgca aacatctatc
     6481 aagagacagg atcagcagga ggctttcgca tgagtattca acatttccgt gtcgccctta
     6541 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     6601 taaaagatgc tgaagatcag ttgggtgcgc gagtgggtta catcgaactg gatctcaaca
     6661 gcggtaagat ccttgagagt tttcgccccg aagaacgctt tccaatgatg agcactttta
     6721 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     6781 gccgcataca ctattctcag aatgacttgg ttgagtattc accagtcaca gaaaagcatc
     6841 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6901 ctgcggccaa cttacttctg acaacgattg gaggaccgaa ggagctaacc gcttttttgc
     6961 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     7021 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacc ttgcgtaaac
     7081 tattaactgg cgaactactt actctagctt cccggcaaca gttgatagac tggatggagg
     7141 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     7201 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     7261 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     7321 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ccgattctag
     7381 gtgcattggc gcagaaaaaa atgcctgatg cgacgctgcg cgtcttatac tcccacatat
     7441 gccagattca gcaacggata cggcttcccc aacttgccca cttccatacg tgtcctcctt
     7501 accagaaatt tatccttaag atcccgaatc gtttaaac
//
