LOCUS       sequence-438878-        8033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SPNS1(E164K) codon-optimized orf with n-terminal 3xFlag GFP in 
            pMXs-IRES-Blast vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8033)
  AUTHORS   Scharenberg SG, Dong W, Ghoochani A, Nyame K, Levin-Konigsberg R, 
            Krishnan AR, Rawat ES, Spees K, Bassik MC, Abu-Remaileh M
  TITLE     An SPNS1-dependent lysosomal lipid transport pathway that enables 
            cell survival under choline limitation.
  JOURNAL   Sci Adv. 2023 Apr 21;9(16):eadf8966. doi: 10.1126/sciadv.adf8966. 
            Epub 2023 Apr 19.
  PUBMED    37075117
REFERENCE   2  (bases 1 to 8033)
  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: "Sci Adv."; 
            date: "2023-04-21"; volume: "9(16):eadf8966. doi"; pages: " 
            10.1126/sciadv.adf8966. Epub 2023 Apr 19"
COMMENT     Sequence Label: pMXs-IRES-blast GFP-SPNS1(E164K)
FEATURES             Location/Qualifiers
     source          1..8033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(259..363)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             393..986
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    1049..1406
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1465..1881
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1811..1833
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1849..1865
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1891..2265
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     2203..2219
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      2294..2303
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2303..2368
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(2402..2404,2405..3115)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2402..2404,2405..3115)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2444..2465)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2705..2724)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3052..3073
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(4756..5330,5331..5333,5334..5342)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4923..4940)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5150..5169
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5343..5738
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MVPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             5909..6378
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(6508..6525)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6679..7267)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6759..6778)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7431..8033,1..189,190..258))
                     /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"
ORIGIN
        1 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
       61 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
      121 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
      181 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
      241 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
      301 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      361 gcgcacattt ccccgaaaag tgccacctgc ataatgaaag accccacctg taggtttggc
      421 aagctagctt aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa
      481 aagttcagat caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct
      541 gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg
      601 ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt
      661 ccccagatgc ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc
      721 cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc
      781 ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg
      841 cgcgccagtc ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt
      901 gcagttgcat ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga
      961 ctacccgtca gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc
     1021 agggaccacc gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg
     1081 attgtctagt gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc
     1141 tctgtatctg gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg
     1201 ggagacgtcc cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc
     1261 gatcgttttg gactctttgg tgcacccccc taataggagg gatatgtggt tctggtagga
     1321 gacgagaacc taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga
     1381 agccgcgccg cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg
     1441 tttctgtatt tgtctgaaaa ttagggccag actgttacca ctcccttaag tttgacctta
     1501 ggtcactgga aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga
     1561 cgttgggtta ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac
     1621 ggcaccttta accgagacct catcacccag gttaagatca aggtcttttc acctggcccg
     1681 catggacacc cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc
     1741 cctccctggg tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc
     1801 ccgtctctcc cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc
     1861 ctcactcctt ctctaggcgc ccccatatgg ccatatgaga tcttatatgg ggcacccccg
     1921 ccccttgtaa acttccctga ccctgacatg acaagagtta ctaacagccc ctctctccaa
     1981 gctcacttac aggctctcta cttagtccag cacgaagtct ggagacctct ggcggcagcc
     2041 taccaagaac aactggaccg accggtggta cctcaccctt accgagtcgg cgacacagtg
     2101 tgggtccgcc gacaccagac taagaaccta gaacctcgct ggaaaggacc ttacacagtc
     2161 ctgctgacca cccccaccgc cctcaaagta gacggcatcg cagcttggat acacgccgcc
     2221 cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta gactgccgga tctagctagt
     2281 taattaagga tccgccacca tggactacaa ggaccacgac ggtgactaca aggaccacga
     2341 catcgactac aaggacgacg acgacaaggg gcatggcacc ggcagcaccg gcagcgaatt
     2401 cgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     2461 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     2521 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     2581 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     2641 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     2701 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     2761 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     2821 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     2881 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2941 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     3001 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     3061 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggagg
     3121 gagcggcccg cggctcgaga tggccggctc tgacaccgcc cctttcctga gccaggccga
     3181 cgatccagac gatggaccag tgccaggaac ccctggactg ccaggctcta caggcaaccc
     3241 caagagcgag gagccagagg tgcctgacca ggagggactg cagaggatca caggactgtc
     3301 ccctggccgg tctgccctga tcgtggccgt gctgtgctac atcaacctgc tgaattatat
     3361 ggatcgcttt accgtggcag gcgtgctgcc agacatcgag cagttcttta acatcggcga
     3421 tagctcctct ggcctgatcc agacagtgtt catcagctcc tacatggtgc tggcccccgt
     3481 gttcggctat ctgggcgatc gctacaatcg gaagtatctg atgtgcggcg gcatcgcctt
     3541 ctggagcctg gtgaccctgg gctctagctt catcccaggc gagcactttt ggctgctgct
     3601 gctgacaagg ggactggtgg gagtgggaaa ggcaagctac tccaccatcg cacctacact
     3661 gatcgccgac ctgtttgtgg ccgatcagag aagcaggatg ctgtccatct tctactttgc
     3721 catcccagtg ggaagcggac tgggatatat cgcaggatcc aaggtgaagg acatggccgg
     3781 cgattggcac tgggcactga gagtgacccc tggactggga gtggtggccg tgctgctgct
     3841 gttcctggtg gtgagagagc cacctagggg agcagtggag aggcactctg acctgccacc
     3901 cctgaaccca acaagctggt gggcagatct gcgcgccctg gcaaggaatc catccttcgt
     3961 gctgtcctct ctgggcttca ccgccgtggc ctttgtgaca ggatccctgg ccctgtgggc
     4021 acctgccttt ctgctgcgct ctagggtggt gctgggagag acccctccat gcctgccagg
     4081 cgactcctgt agctcctctg attctctgat ctttggcctg atcacctgcc tgacaggcgt
     4141 gctgggagtg ggactgggag tggagatcag ccggagactg agacactcca acccaagggc
     4201 agacccactg gtgtgcgcaa ccggactgct gggctccgcc cccttcctgt ttctgtctct
     4261 ggcatgtgca aggggaagca tcgtggcaac ctacatcttc atctttatcg gcgagacact
     4321 gctgagcatg aattgggcca tcgtggccga tatcctgctg tatgtggtca tcccaaccag
     4381 gcgctccaca gccgaggcct tccagatcgt gctgtctcac ctgctgggcg acgcaggaag
     4441 cccatatctg atcggcctga tctccgatcg cctgcggaga aattggcccc cttctttcct
     4501 gagcgagttt cgggccctgc agttcagcct gatgctgtgc gcctttgtgg gcgccctggg
     4561 aggagcagcc ttcctgggca ccgccatctt tatcgaggcc gacaggcgcc gggcacagct
     4621 gcacgtgcag ggactgctgc acgaggcagg ctccaccgac gatagaatcg tggtgcccca
     4681 gagaggcagg tccacaaggg tgcctgtggc ctctgtgctg atctgagcgg ccgctacgta
     4741 aattccgccc ccccccccct ctccctcccc cccccctaac gttactggcc gaagccgctt
     4801 ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg
     4861 caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc
     4921 ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga
     4981 agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc
     5041 tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc
     5101 acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc
     5161 aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga
     5221 tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg
     5281 ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa
     5341 ccatggttcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg gctacaatca
     5401 acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct agcgacggcc
     5461 gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca gaactcgtgg
     5521 tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa
     5581 atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt ctcgatctgc
     5641 atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca gttgggattc
     5701 gtgaattgct gccctctggt tatgtgtggg agggctaagt cgacgataaa ataaaagatt
     5761 ttatttagtc tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta
     5821 gcttaagtaa cgccattttg caaggcatgg aaaaatacat aactgagaat agagaagttc
     5881 agatcaaggt caggaacaga tggaacagct gaatatgggc caaacaggat atctgtggta
     5941 agcagttcct gccccggctc agggccaaga acagatggaa cagctgaata tgggccaaac
     6001 aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag
     6061 atgcggtcca gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag
     6121 gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt
     6181 tcgcgcgctt ctgctccccg agctcaataa aagagcccac aacccctcac tcggggcgcc
     6241 agtcctccga ttgactgagt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt
     6301 gcatccgact tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc
     6361 gtcagcgggg gtctttcaca tgcagcatgt atcaaaatta atttggtttt ttttcttaag
     6421 tatttacatt aaatggccat agttgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6481 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     6541 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     6601 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     6661 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     6721 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     6781 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     6841 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6901 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6961 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     7021 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     7081 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     7141 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     7201 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     7261 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     7321 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttgcg
     7381 gccggccgca aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     7441 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     7501 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     7561 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     7621 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     7681 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     7741 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     7801 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     7861 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     7921 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     7981 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctc
//
