LOCUS       sequence-182684-        6395 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  selectable HDR vector to introduce K700E mutation within mus Sf3b1 
            gene (for use with sgSf3b1(T1) and pL452(hygro)-Sf3b1-K700K plasmids
            enabling generation of hemizygous K700E mutation).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6395)
  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 6395)
  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-Sf3b1-K700E
FEATURES             Location/Qualifiers
     source          1..6395
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(16..515)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(64..83)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     457..480
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    complement(580..613)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(1496..1514)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1535..1551)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1535..1551)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1548..1570)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1559..1575
                     /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(1583..1589,1590..1607,1608..1613))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1628..1649
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1766..1783)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1937..2525)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2017..2036)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2696..3487,3488..3556))
                     /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     3319..3338
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3557..3661)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3688..4143
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3775..3794)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3985..4006
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4269..4291
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4283..4300
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4284..4300
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4307..4326
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4307..4325
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(5178..5211)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    5268..5492
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5481..5498
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(5530..6333)
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5641..5660)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6251..6270
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6352..6395,1..4))
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
ORIGIN
        1 caacaggctg caggtcgaaa ggcccggaga tgaggaagag gagaacagcg cggcagacgt
       61 gcgcttttga agcgtgcaga atgccgggcc tccggaggac cttcgggcgc ccgccccgcc
      121 cctgagcccg cccctgagcc cgcccccgga cccacccctt cccagcctct gagcccagaa
      181 agcgaaggag caaagctgct attggccgct gccccaaagg cctacccgct tccattgctc
      241 agcggtgctg tccatctgca cgagactagt gagacgtgct acttccattt gtcacgtcct
      301 gcacgacgcg agctgcgggg cgggggggaa cttcctgact aggggaggag tagaaggtgg
      361 cgcgaagggg ccaccaaaga acggagccgg ttggcgccta ccggtggatg tggaatgtgt
      421 gcgaggccag aggccacttg tgtagcgcca agtgcccagc ggggctgcta aagcgcatgc
      481 tccagactgc cttgggaaaa gcgcctcccc tacccggtag aatttcgacg acctgcagcc
      541 aagctagctt ggctggacgt aaactcctct tcagacctaa taacttcgta tagcatacat
      601 tatacgaagt tatattaagg gttattgaat atgatcggaa ttgggctgca ggaattctac
      661 tagcaacaaa aactgatcta atgagcgatt cacctttcct ctgtgttggc ggataccttt
      721 ccaaagcggt ttcaggacag aatcaaaaga ctcaatacca taaggagttg ctgcctcagc
      781 caaagcagca atggccagag cactgatggt ccgaacttcc tgctgctcat ccacgagacc
      841 tatacaatca gacatggtta tgagtaggta cccacaccta attttcataa tacaatccaa
      901 attttagcaa tgtatctaac catgtatcca aactgcaata atttttttat agtacataaa
      961 atttaggcaa tggtaatttg ttaacagaaa atattacaaa ccccatttca aaatttgcgt
     1021 gagaagtctt tcatttaatt acacatacac ctctccccat caggacagct gatctataaa
     1081 agatacacca cttaccatgt tcaatgattt caaccaaact tctgagatgt ggcaagatag
     1141 cacagcccat gagaatggct atctgttgta caatcttaat accagtgtgc ctcgcttgcc
     1201 aagacttctt gcttttgcac acagctttta agaaaggcaa taaagaaggg atgcccaggg
     1261 cagaggctac gacagcaaaa gctctagctg ttgtgtttcg gacatattca tccatgttat
     1321 ctatatcagg tctcatggta gaaatcatag tagccagacc agcagcctaa agagaaaagt
     1381 gacaacagcc acttaagtta gtaccattaa tcctcaaccc atcaaaagac aaacttcctt
     1441 ttctaggaat ttaatgtgtc gacctcgagg gggggcccgg taccagcttt tgttcccttt
     1501 agtgagggtt aatttcgagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     1561 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     1621 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1681 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1741 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1801 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1861 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1921 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1981 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2041 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2101 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2161 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2221 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2281 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2341 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2401 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2461 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2521 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2581 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2641 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2701 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2761 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2821 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     2881 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2941 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3001 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3061 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3121 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3181 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3241 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3301 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3361 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3421 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3481 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3541 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     3601 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3661 gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg
     3721 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3781 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3841 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3901 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3961 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     4021 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     4081 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     4141 tttccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     4201 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     4261 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact
     4321 atagggcgaa ttggagctcc accgcggtgg cggccgccag agtgatttct aggctactta
     4381 gtgagaccct atctcagaca gacagaccaa acagaaagaa tggactttaa aaattccatc
     4441 tcttgtcaca gtaaagtaca tacaagtata aattttaaga cagtcaaagc aaattatagc
     4501 atgtaatatg cattctaagt aggtaaagaa ttagtcaaaa agatttatga gaatcaaagt
     4561 ttagcaataa aaggtcacct gtcggtaatt tcttctatcc aaagccattc tgtgttgcca
     4621 aaagtgttta aagaaaggtg gaagaatctc tgttttaatg tagtttgctt ctacaccatc
     4681 agttccacaa cactgtttca ccacctataa ggttaataaa aaagaaatat atatatcaaa
     4741 tgcagcactg gcaaaacaca aaacaatcca acacagttat gagtaggtac caatacctaa
     4801 ttttcgtaat gcaatccaaa ttttagcaat gtatctaacc atgtatccaa gctgtaataa
     4861 cttttacagt acaaaaatag gtttgatttg tttttaatgt tataaagatc tctctgaact
     4921 tcctaaaaca tgcacatacc cacaaactga gcaactaatt acttaccttc aaaacaattt
     4981 tcttcatttc ttcatcagga gactggaatt ctcgaatgag gattaacatc acttctctag
     5041 tatagtagtt ggcatattct gcatccataa gaggaataag atacccaata gccttcaaga
     5101 aagcagccag accctattta aaaatatata cgttagtaaa ttaagtttcg gttatctgga
     5161 tcccctcgag ggacctaata acttcgtata gcatacatta tacgaagtta tattaagggt
     5221 tccgcaagct ctagtcgagc cccagctggt tctttccgcc tcagaagcca tagagcccac
     5281 cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt cctgccccac
     5341 cccacccccc agaatagaat gacacctact cagacaatgc gatgcaattt cctcatttta
     5401 ttaggaaagg acagtgggag tggcaccttc cagggtcaag gaaggcacgg gggaggggca
     5461 aacaacagat ggctggcaac tagaaggcac agtcgaggct gatcagcgag ctctagagaa
     5521 ttgatcccct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg
     5581 agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc
     5641 aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca
     5701 gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc
     5761 atgggtcacg acgagatcat cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc
     5821 ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc
     5881 catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc
     5941 cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg
     6001 agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct
     6061 tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca
     6121 cgatagccgc gctgcctcgt cctgcagttc attcagggca ccggacaggt cggtcttgac
     6181 aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat
     6241 tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc
     6301 gtgcaatcca tcttgttcaa tggccgatcc catggtttag ttcctcacct tgtcgtatta
     6361 tactatgccg atatactatg ccgatgatta attgt
//
