LOCUS       sequence-438870-        7295 bp    DNA     circular SYN 14-APR-2025
DEFINITION  SPNS1(E164K) codon-optimized orf with n-terminal 3xFlag in 
            pMXs-IRES-Blast vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7295)
  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 7295)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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 3xFlag-SPNS1(E164K)
FEATURES             Location/Qualifiers
     source          1..7295
                     /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"
     misc_feature    join(4018..4592,4593..4595,4596..4604)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4185..4202)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4412..4431
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4605..5000
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MVPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             5171..5640
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5770..5787)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5941..6529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6021..6040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6693..7295,1..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 catggccggc tctgacaccg cccctttcct gagccaggcc gacgatccag acgatggacc
     2461 agtgccagga acccctggac tgccaggctc tacaggcaac cccaagagcg aggagccaga
     2521 ggtgcctgac caggagggac tgcagaggat cacaggactg tcccctggcc ggtctgccct
     2581 gatcgtggcc gtgctgtgct acatcaacct gctgaattat atggatcgct ttaccgtggc
     2641 aggcgtgctg ccagacatcg agcagttctt taacatcggc gatagctcct ctggcctgat
     2701 ccagacagtg ttcatcagct cctacatggt gctggccccc gtgttcggct atctgggcga
     2761 tcgctacaat cggaagtatc tgatgtgcgg cggcatcgcc ttctggagcc tggtgaccct
     2821 gggctctagc ttcatcccag gcgagcactt ttggctgctg ctgctgacaa ggggactggt
     2881 gggagtggga aaggcaagct actccaccat cgcacctaca ctgatcgccg acctgtttgt
     2941 ggccgatcag agaagcagga tgctgtccat cttctacttt gccatcccag tgggaagcgg
     3001 actgggatat atcgcaggat ccaaggtgaa ggacatggcc ggcgattggc actgggcact
     3061 gagagtgacc cctggactgg gagtggtggc cgtgctgctg ctgttcctgg tggtgagaga
     3121 gccacctagg ggagcagtgg agaggcactc tgacctgcca cccctgaacc caacaagctg
     3181 gtgggcagat ctgcgcgccc tggcaaggaa tccatccttc gtgctgtcct ctctgggctt
     3241 caccgccgtg gcctttgtga caggatccct ggccctgtgg gcacctgcct ttctgctgcg
     3301 ctctagggtg gtgctgggag agacccctcc atgcctgcca ggcgactcct gtagctcctc
     3361 tgattctctg atctttggcc tgatcacctg cctgacaggc gtgctgggag tgggactggg
     3421 agtggagatc agccggagac tgagacactc caacccaagg gcagacccac tggtgtgcgc
     3481 aaccggactg ctgggctccg cccccttcct gtttctgtct ctggcatgtg caaggggaag
     3541 catcgtggca acctacatct tcatctttat cggcgagaca ctgctgagca tgaattgggc
     3601 catcgtggcc gatatcctgc tgtatgtggt catcccaacc aggcgctcca cagccgaggc
     3661 cttccagatc gtgctgtctc acctgctggg cgacgcagga agcccatatc tgatcggcct
     3721 gatctccgat cgcctgcgga gaaattggcc cccttctttc ctgagcgagt ttcgggccct
     3781 gcagttcagc ctgatgctgt gcgcctttgt gggcgccctg ggaggagcag ccttcctggg
     3841 caccgccatc tttatcgagg ccgacaggcg ccgggcacag ctgcacgtgc agggactgct
     3901 gcacgaggca ggctccaccg acgatagaat cgtggtgccc cagagaggca ggtccacaag
     3961 ggtgcctgtg gcctctgtgc tgatctgagc ggccgctacg taaattccgc cccccccccc
     4021 ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc
     4081 gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa
     4141 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat
     4201 gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac
     4261 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg
     4321 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt
     4381 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg
     4441 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac
     4501 atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga
     4561 cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggtt cctttgtctc
     4621 aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg
     4681 aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca
     4741 atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg
     4801 ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct
     4861 tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca
     4921 tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg
     4981 gttatgtgtg ggagggctaa gtcgacgata aaataaaaga ttttatttag tctccagaaa
     5041 aaggggggaa tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt
     5101 tgcaaggcat ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca
     5161 gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc
     5221 tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc
     5281 agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag
     5341 cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt
     5401 gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc
     5461 cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga
     5521 gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct
     5581 cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca
     5641 catgcagcat gtatcaaaat taatttggtt ttttttctta agtatttaca ttaaatggcc
     5701 atagttgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     5761 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5821 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5881 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5941 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6001 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6061 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6121 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6181 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6241 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6301 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6361 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6421 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6481 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6541 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6601 catgagatta tcaaaaagga tcttcaccta gatccttttg cggccggccg caaatcaatc
     6661 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6721 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6781 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6841 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6901 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6961 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     7021 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7081 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7141 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7201 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7261 ttctgagaat agtgtatgcg gcgaccgagt tgctc
//
