LOCUS       sequence-190769-        6631 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  selectable HDR vector to introduce silent mutation (position 700) in
            mus Sf3b1 gene (for use with pL452-Sf3b1-K700E and sgSf3b1(T1) gRNA 
            vectors enabling generation of hemizygous K700E mutation).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6631)
  AUTHORS   Gupta AK, Murthy T, Paul KV, Ramirez O, Fisher JB, Rao S, Rosenberg 
            AB, Seelig G, Minella AC, Pillai MM
  TITLE     Degenerate minigene library analysis enables identification of 
            altered branch point utilization by mutant splicing factor 3B1 
            (SF3B1).
  JOURNAL   Nucleic Acids Res. 2018 Nov 20. pii: 5193340. doi: 
            10.1093/nar/gky1161.
  PUBMED    30462273
REFERENCE   2  (bases 1 to 6631)
  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. 2018 Nov 20. pii: 5193340. doi: 10.1093/nar/gky1161."
COMMENT     Sequence Label: pL452(hygro)-Sf3b1-K700K
FEATURES             Location/Qualifiers
     source          1..6631
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     gap             133..147
                     /estimated_length=15
     primer_bind     192..211
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        271..318
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             363..1382
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     primer_bind     complement(1408..1425)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1414..1638
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    1695..1728
                     /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(2580..2599)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2581..2599)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2606..2623)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2606..2622)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2615..2637)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2763..3218)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2900..2921)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3112..3131
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3245..3349
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3350..3418,3419..4210)
                     /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(3568..3587)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4381..4969
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4870..4889
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5123..5140
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5257..5278
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5293..5298,5299..5316,5317..5323)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5331..5347
                     /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     5336..5358
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5355..5371
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5355..5371
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5392..5410
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    6293..6326
                     /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(6426..6449)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
ORIGIN
        1 tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg
       61 gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg
      121 tgggtccggg ggnnnnnnnn nnnnnnngct caggggcggg gcgggcgccc gaaggtcctc
      181 cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc
      241 ctcatctccg ggcctttcga cctgcagcct gttgacaatt aatcatcggc atagtatatc
      301 ggcatagtat aatacgacaa ggtgaggaac taaaccaagc ttccaccatg gatagatccg
      361 gaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
      421 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
      481 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt
      541 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg
      601 agttcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag
      661 acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga
      721 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg
      781 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact
      841 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga
      901 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca
      961 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt
     1021 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta
     1081 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc
     1141 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca
     1201 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg
     1261 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg
     1321 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     1381 agcatcgatc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     1441 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     1501 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     1561 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     1621 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg actagagctt
     1681 gcggaaccct taatataact tcgtataatg tatgctatac gaagttatta ggtccctcga
     1741 ggggatccag ataaccgaaa cttaatttac taacgtatat atttttaaat agggtctggc
     1801 tgctttcttg aaggctattg ggtatcttat tcctcttatg gatgcagaat atgccaacta
     1861 ctatactaga gaagtgatgt taatcctcat tcgagaattt cagtctcctg atgaagaaat
     1921 gaagaaaatt gttttgaagg taagtaatta gttgctcagt ttgtgggtat gtgcatgttt
     1981 taggaagttc agagagatct ttataacatt aaaaacaaat caaacctatt tttgtactgt
     2041 aaaagttatt acagcttgga tacatggtta gatacattgc taaaatttgg attgcattac
     2101 gaaaattagg tattggtacc tactcataac tgtgttggat tgttttgtgt tttgccagtg
     2161 ctgcatttga tatatatatt tcttttttat taaccttata ggtggtgaaa cagtgttgtg
     2221 gaactgatgg tgtagaagca aactacatta aaacagagat tcttccacct ttctttaaac
     2281 acttttggca acacagaatg gctttggata gaagaaatta ccgacaggtg accttttatt
     2341 gctaaacttt gattctcata aatctttttg actaattctt tacctactta gaatgcatat
     2401 tacatgctat aatttgcttt gactgtctta aaatttatac ttgtatgtac tttactgtga
     2461 caagagatgg aatttttaaa gtccattctt tctgtttggt ctgtctgtct gagatagggt
     2521 ctcactaagt agcctagaaa tcactctggc ggccgccacc gcggtggagc tccaattcgc
     2581 cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     2641 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     2701 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     2761 ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     2821 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     2881 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     2941 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     3001 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     3061 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     3121 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     3181 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     3241 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3301 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     3361 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     3421 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     3481 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     3541 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     3601 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3661 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3721 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3781 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3841 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     3901 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3961 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     4021 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     4081 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     4141 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     4201 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     4261 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4321 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4381 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4441 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4501 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4561 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4621 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4681 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4741 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4801 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4861 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4921 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4981 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5041 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     5101 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     5161 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     5221 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
     5281 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     5341 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctcg aaattaaccc
     5401 tcactaaagg gaacaaaagc tggtaccggg ccccccctcg aggtcgacac attaaattcc
     5461 tagaaaagga agtttgtctt ttgatgggtt gaggattaat ggtactaact taagtggctg
     5521 ttgtcacttt tctctttagg ctgctggtct ggctactatg atttctacca tgagacctga
     5581 tatagataac atggatgaat atgtccgaaa cacaacagct agagcttttg ctgtcgtagc
     5641 ctctgccctg ggcatccctt ctttattgcc tttcttaaaa gctgtgtgca aaagcaagaa
     5701 gtcttggcaa gcgaggcaca ctggtattaa gattgtacaa cagatagcca ttctcatggg
     5761 ctgtgctatc ttgccacatc tcagaagttt ggttgaaatc attgaacatg gtaagtggtg
     5821 tatcttttat agatcagctg tcctgatggg gagaggtgta tgtgtaatta aatgaaagac
     5881 ttctcacgca aattttgaaa tggggtttgt aatattttct gttaacaaat taccattgcc
     5941 taaattttat gtactataaa aaaattattg cagtttggat acatggttag atacattgct
     6001 aaaatttgga ttgtattatg aaaattaggt gtgggtacct actcataacc atgtctgatt
     6061 gtataggtct cgtggatgag cagcagaagg ttcggaccat cagtgctctg gccattgctg
     6121 ctttggctga ggcagcaact ccttatggta ttgagtcttt tgattctgtc ctgaaaccgc
     6181 tttggaaagg tatccgccaa cacagaggaa aggtgaatcg ctcattagat cagtttttgt
     6241 tgctagtaga attcctgcag cccaattccg atcatattca ataaccctta atataacttc
     6301 gtataatgta tgctatacga agttattagg tctgaagagg agtttacgtc cagccaagct
     6361 agcttggctg caggtcgtcg aaattctacc gggtagggga ggcgcttttc ccaaggcagt
     6421 ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc
     6481 ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct
     6541 tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt
     6601 cgtgcaggac gtgacaaatg gaagtagcac g
//
