LOCUS       sequence-236037-        7674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sphingosine kinase 2, which catalyzes the phosphorylation 
            of sphingosine to form sphingosine 1-phosphate in mice.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7674)
  AUTHORS   Kharel Y, Agah S, Huang T, Mendelson AJ, Eletu OT, Barkey-Bircann P,
            Gesualdi J, Smith JS, Santos WL, Lynch KR
  TITLE     Saccharomyces cerevisiae as a platform for assessing sphingolipid 
            lipid kinase inhibitors.
  JOURNAL   PLoS One. 2018 Apr 19;13(4):e0192179. doi: 
            10.1371/journal.pone.0192179. eCollection 2018.
  PUBMED    29672528
REFERENCE   2  (bases 1 to 7674)
  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: "PLoS One.";
            date: "2018-04-19"; volume: "13(4):e0192179. doi"; pages: " 
            10.1371/journal.pone.0192179. eCollection 2018"
COMMENT     Sequence Label: MmSphk2
FEATURES             Location/Qualifiers
     source          1..7674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        22..463
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    22..139
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     434..457
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     495..514
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        495..513
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             533..556
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      2443..2690
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(2459..2477)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(2767..2784)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2938..3526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3018..3037)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3697..4488,4489..4557))
                     /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     4320..4339
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(4653..5456)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(5457..5672)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     rep_origin      6273..7150
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     rep_origin      complement(7161..7674)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7356..7377)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7568..7587
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ggggatcgat ccactagtcc acggattaga agccgccgag cgggtgacag ccctccgaag
       61 gaagactctc ctccgtgcgt cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc
      121 ctcgcgccgc actgctccga acaataaaga ttctacaata ctagctttta tggttatgaa
      181 gaggaaaaat tggcagtaac ctggccccac aaaccttcaa atgaacgaat caaattaaca
      241 accataggat gataatgcga ttagtttttt agccttattt ctggggtaat taatcagcga
      301 agcgatgatt tttgatctat taacagatat ataaatgcaa aaactgcata accactttaa
      361 ctaatacttt caacattttc ggtttgtatt acttcttatt caaatgtaat aaaagtatca
      421 acaaaaaatt gttaatatac ctctatactt taacgtcaag gagaaaaaac cccggatcgg
      481 actactagca gctgtaatac gactcactat agggaatatt aagcttacca tggactacaa
      541 ggacgatgac gataagggat ctatggcccc accaccacta ctgccagtgg ctgccagcac
      601 tccaatcctg cacggcgagt ttggttccta cccggccaac ggcccacggt ttgccctcac
      661 cctcacaaca caagccctac acatacagcg actacgccca aagccagaag cccggccccg
      721 agatggtcta gtctctctgg atgaggtctc gggctgtggc accctgcaga gccgtagccc
      781 cgaggacact gcagcctact tctgcatcta cacctaccca cgtggccgtc gagggggccg
      841 gcgcagagct acgcggacct tccgggcgga tggggccacc acttatgagg agaatcgtgc
      901 agaggcccag cgctgggcca ctgccctcac gtgtctcctc cgaggagtgc ctctgtcagg
      961 ggaccaggaa atcacccctg aattgctgcc ccggaagccc aggctgctca tattggtcaa
     1021 tccctttggg gggcggggcc tggcctggca gcgctgtatg gaccacgtgg tgccaatgat
     1081 ctctgaagct gggctgtcct tcaacctcat acagacagaa cgacagaacc atgcccgtga
     1141 gctggtgcag gggttaagcc tgagtgagtg ggaaggcatt gtcactgtgt ctggagacgg
     1201 gctgctttac gaggtgctga atgggctcct tgatcggcca gactgggagg atgccgtgcg
     1261 gatgcccatt ggtgtcctcc cctgtggatc gggcaatgcg ctagctgggg cggtgaacca
     1321 tcatggcggg tttgagcagg ttgtcggtgt tgacctgttg ctcaactgct cgcttcttct
     1381 ctgccgtggt ggcagccatc ctctggacct gctctctgtg acgctagcct cgggatcccg
     1441 ctgtttttcc ttcctgtcag tggcctgggg attcttgtca gatgtggaca ttcacagtga
     1501 gcgcttcagg gccctgggca gcgctcgatt cacactgggt gcagtgctag gcctggcctc
     1561 gttgcatacc taccgtggac gcctctccta cctccccgct accacagaac cagccttgcc
     1621 catcccaggc cacagtctgc ctcgagccaa gtcagaacta gtcttggctc cagccccagc
     1681 ccccgccgcc acccactcgc ctctacatcg atctgtgtct gacctgcctc tgccccttcc
     1741 ccagcctgcc ttggtctccc ctggctcccc tgagcccctg ccggacctgt ccctcaatgg
     1801 tggtggtcca gagctgactg gagactgggg aggagctggg gatgcacctc tgtccccaga
     1861 cccactgctg ccttcatccc ccaacgctct caaaacagct cagctttcac ccatcgctga
     1921 agggccccca gaaatgccag catcttcggg gttcctgcct cccacccaca gtgccccaga
     1981 agcctctacc tggggcccag tggaccacct cctccctccc ctgggctctc cactgcccca
     2041 agactgggtg acaatagagg gggagtttgt actcatgttg ggcatcttgc cgagccacct
     2101 ctgcgcagac ctgatggcag ccccacatgc acgctttgat gatggcgttg tgcacctgtg
     2161 ttgggtgcgg agcggcatct cacgggctgc acttctacgc attttgctgg ccatggagca
     2221 tggaaaccac ttcagcctgg gctgccccca tctgggctat gctgcagcac gtgccttccg
     2281 ccttgaacca ctcacgcctc gtggcctgct cactgtagat ggggagttag tggagtatgg
     2341 gccaatacag gcgcaggtgc acccaggtct cgccacgctg ctcactgggc ctgcaggtca
     2401 aaagccacaa gcctgagtcg agcatgcatc tagagggccg catcatgtaa ttagttatgt
     2461 cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac
     2521 aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat
     2581 ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac
     2641 tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc aagctgcggc
     2701 cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     2761 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     2821 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2881 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2941 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3001 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3061 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3121 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3181 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3241 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3301 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3361 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3421 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3481 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3541 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3601 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3661 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3721 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     3781 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     3841 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     3901 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     3961 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4021 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4081 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4141 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4201 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4261 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4321 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4381 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4441 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4501 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4561 cttccttttt caatgggtaa taactgatat aattaaattg aagctctaat ttgtgagttt
     4621 agtatacatg catttactta taatacagtt ttttagtttt gctggccgca tcttctcaaa
     4681 tatgcttccc agcctgcttt tctgtaacgt tcaccctcta ccttagcatc ccttcccttt
     4741 gcaaatagtc ctcttccaac aataataatg tcagatcctg tagagaccac atcatccacg
     4801 gttctatact gttgacccaa tgcgtctccc ttgtcatcta aacccacacc gggtgtcata
     4861 atcaaccaat cgtaaccttc atctcttcca cccatgtctc tttgagcaat aaagccgata
     4921 acaaaatctt tgtcgctctt cgcaatgtca acagtaccct tagtatattc tccagtagat
     4981 agggagccct tgcatgacaa ttctgctaac atcaaaaggc ctctaggttc ctttgttact
     5041 tcttctgccg cctgcttcaa accgctaaca atacctgggc ccaccacacc gtgtgcattc
     5101 gtaatgtctg cccattctgc tattctgtat acacccgcag agtactgcaa tttgactgta
     5161 ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa aattgtactt ggcggataat
     5221 gcctttagcg gcttaactgt gccctccatg gaaaaatcag tcaagatatc cacatgtgtt
     5281 tttagtaaac aaattttggg acctaatgct tcaactaact ccagtaattc cttggtggta
     5341 cgaacatcca atgaagcaca caagtttgtt tgcttttcgt gcatgatatt aaatagcttg
     5401 gcagcaacag gactaggatg agtagcagca cgttccttat atgtagcttt cgacatgatt
     5461 tatcttcgtt tcctgcaggt ttttgttctg tgcagttggg ttaagaatac tgggcaattt
     5521 catgtttctt caacactaca tatgcgtata tataccaatc taagtctgtg ctccttcctt
     5581 cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat ttcaaggaaa ccgaaatcaa
     5641 aaaaaagaat aaaaaaaaaa tgatgaattg aaaagctagc ttatcgatga taagctgtca
     5701 aacatgagaa ttaattccac ggactataga ctatacctag tatactccgt ctactgtacg
     5761 atacacttcc gctcaggtcc ttgtccttta acgaggcctt accactcttt tgttactcta
     5821 ttgatccagc tcagcaaagg cagtgtgatc taagattcta tcttcgcgat gtagtaaaac
     5881 tagctagacc gagaaagaga ctagaaatgc aaaaggcact tctacaatgg ctgccatcat
     5941 tattatccga tgtgacgctg cagcttctca atgatattcg aatacgcttt gaggagatac
     6001 agcctaatat ccgacaaact gttttacaga tttacgatcg tacttgttac ccatcattga
     6061 attttgaaca tccgaacctg ggagttttcc ctgaaacaga tagtatattt gaacctgtat
     6121 aataatatat agtctagcgc tttacggaag acaatgtatg tatttcggtt cctggagaaa
     6181 ctattgcatc tattgcatag gtaatcttgc acgtcgcatc cccggttcat tttctgcgtt
     6241 tccatcttgc acttcaatag catatctttg ttaacgaagc atctgtgctt cattttgtag
     6301 aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta
     6361 cagaacagaa atgcaacgcg aaagcgctat tttaccaacg aagaatctgt gcttcatttt
     6421 tgtaaaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat
     6481 ttttacagaa cagaaatgca acgcgagagc gctattttac caacaaagaa tctatacttc
     6541 ttttttgttc tacaaaaatg catcccgaga gcgctatttt tctaacaaag catcttagat
     6601 tacttttttt ctcctttgtg cgctctataa tgcagtctct tgataacttt ttgcactgta
     6661 ggtccgttaa ggttagaaga aggctacttt ggtgtctatt ttctcttcca taaaaaaagc
     6721 ctgactccac ttcccgcgtt tactgattac tagcgaagct gcgggtgcat tttttcaaga
     6781 taaaggcatc cccgattata ttctataccg atgtggattg cgcatacttt gtgaacagaa
     6841 agtgatagcg ttgatgattc ttcattggtc agaaaattat gaacggtttc ttctattttg
     6901 tctctatata ctacgtatag gaaatgttta cattttcgta ttgttttcga ttcactctat
     6961 gaatagttct tactacaatt tttttgtcta aagagtaata ctagagataa acataaaaaa
     7021 tgtagaggtc gagtttagat gcaagttcaa ggagcgaaag gtggatgggt aggttatata
     7081 gggatatagc acagagatat atagcaaaga gatacttttg agcaatgttt gtggaagcgg
     7141 tattcgcaat gggaagctcc accccggttg ataatcagaa aagccccaaa aacaggaaga
     7201 ttgtataagc aaatatttaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     7261 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     7321 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     7381 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     7441 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     7501 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     7561 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     7621 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgt
//
