LOCUS       sequence-435794-        6729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for ceramide (for stable mammalian expression).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6729)
  AUTHORS   Girik V, van Ek L, Dentand Quadri I, Azam M, Cruz Cobo M, Mandavit 
            M, Riezman I, Riezman H, Gavin AC, Nunes-Hasler P
  TITLE     Development of Genetically Encoded Fluorescent KSR1-Based Probes to 
            Track Ceramides during Phagocytosis.
  JOURNAL   Int J Mol Sci. 2024 Mar 5;25(5):2996. doi: 10.3390/ijms25052996.
  PUBMED    38474242
REFERENCE   2  (bases 1 to 6729)
  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: "Int J Mol 
            Sci."; date: "2024-03-5"; volume: "25(5):2996. doi"; pages: " 
            10.3390/ijms25052996"
COMMENT     Sequence Label: pSbi-pur-KSR1-CA3-EGFP (C-KSR)
FEATURES             Location/Qualifiers
     source          1..6729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        109..213
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(214..282,283..1074)
                     /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(432..451)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1245..1833
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1734..1753
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1987..2004
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     2378..2397
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    2396..2451
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     2497..2516
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(2616..3215)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2699..2719)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3196..3215
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        3847..5029
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     primer_bind     4977..4997
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      5079..5088
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    5340..5392
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      5400..5409
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5406..5408,5409..5411,5412..6125)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5406..5408,5409..5411,5412..6122)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5451..5472)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5712..5731)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6059..6080
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6176..6193)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6182..6406
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 aattttaaca aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc
       61 tgtgcggtat ttcacaccgc atcaggtggc acttttcggg gaaatgtgcg cggaacccct
      121 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
      181 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
      241 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
      301 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
      361 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
      421 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
      481 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
      541 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
      601 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
      661 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
      721 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
      781 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
      841 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
      901 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
      961 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     1021 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     1081 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     1141 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     1201 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     1261 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     1321 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     1381 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     1441 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     1501 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     1561 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     1621 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     1681 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     1741 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     1801 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     1861 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     1921 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     1981 gagcgcagcg agtcagtgag cgaggaagcg gatccctata cagttgaagt cggaagttta
     2041 catacacctt agccaaatac atttaaactc actttttcac aattcctgac atttaatcct
     2101 agtaaaaatt ccctgtctta ggtcagttag gatcaccact ttattttaag aatgtgaaat
     2161 atcagaataa tagtagagag aatgattcat ttcagctttt atttctttca tcacattccc
     2221 agtgggtcag aagtttacat acactcgact cctctgcaga atgcggcgat gtttcggtaa
     2281 ggggtcccga gtagctagtt catggcagcc agcatatggc atatgttgcc aaactctaaa
     2341 ccaaatactc attctgatgt tttaaatgat ttgccctccc atatgtcctt ccgagtgaga
     2401 gacacaaaaa attccaacac actattgcaa tgaaaataaa tttcctttat tagccagaag
     2461 tcagatgctc aaggggcttc atgatgtccc cataattttt ggcagaggga aaaagatctc
     2521 agtggtattt gtgagccagg gcattggcca caccagccac caccttctga taggcagcct
     2581 gcacctgagg agtgaattca cgacaggcct tcgaatcagg caccgggctt gcgggtcatg
     2641 caccaggtgc gcggtccttc gggcacctcg acgtcggcgg tgacggtgaa gccgagccgc
     2701 tcgtagaagg ggaggttgcg gggcgcggag gtctccagga aggcgggcac cccggcgcgc
     2761 tcggccgcct ccactccggg gagcacgacg gcgctgccca gacccttgcc ctggtggtcg
     2821 ggcgagacgc cgacggtggc caggaaccac gcgggctcct tgggccggtg cggcgccagg
     2881 aggccttcca tctgttgctg cgcggccagc cgggaaccgc tcaactcggc catgcgcggg
     2941 ccgatctcgg cgaacaccgc ccccgcttcg acgctctccg gcgtggtcca gaccgccacc
     3001 gcggcgccgt cgtccgcgac ccacaccttg ccgatgtcga gcccgacgcg cgtgaggaag
     3061 agttcttgca gctcggtgac ccgctcgatg tggcggtccg gatcgacggt gtggcgcgtg
     3121 gcggggtagt cggcgaacgc ggcggcgagg gtgcgtacgg ccctggggac gtcgtcgcgg
     3181 gtggcgaggc gcaccgtggg cttgtactcg gtcatggtgg cctcaggtgc agaggtttct
     3241 acagggaaag agagaaaggc tacagttagt ttgctgtgat gatgctaagt gccgaggtca
     3301 gatgtctatg gattgtacac ggaatcccaa cgcaggaacc aaaaaacact cacccaaaaa
     3361 aatggcgcta aggccgagag aaaggcgaag ttccgtctac ggctatatac cttgaaacag
     3421 gggcggcaga agtgacgcaa cgcggttgcg cggacggctt cctgtgtttt cattggtcga
     3481 agcgttctgc gcgtgcgcag agattcccca gggaaggcgg gccctcttcc gaatgtccaa
     3541 tccggacatt cttccggttg gacctccgcg gtcagcgctg tgcccggcga gagatcacgt
     3601 ggggcgcgga ggcggtgctg ctggggcacg gccgtccagc ctcggcggcc atatttttga
     3661 ggggctgttc atctcgttca cacgctctgt ccgccatgtt tgtgagtgga agcgccatta
     3721 ccccttcaag cgactgaagg ctgcagggcc tctggtggcc cgcatgggga gaccagaccc
     3781 gccaggcccg cctttccgca ctcagtccgg gcttacttta ttttgtgaga cagggtctcg
     3841 cctagaggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     3901 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
     3961 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     4021 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta
     4081 agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct
     4141 tgaattactt ccacgcccct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg
     4201 aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt
     4261 tgaggcctgg cttgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg
     4321 tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct
     4381 ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt
     4441 ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg
     4501 gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc
     4561 tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg
     4621 gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc
     4681 aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag
     4741 ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag
     4801 gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt
     4861 ttatgcgatg gagtttcccc atactgagtg ggtggagact gaagttaggc cagcttggca
     4921 cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa
     4981 gcctcagaca gtggttcaaa gtttttttct tccatttaag gtgtcgtgaa aactacccca
     5041 agctggcctc tgaggcctga accgtcagat ccgctagcgc caccatgggg aaccgcattg
     5101 atgacgtctc ctcgatgagg tttgatctct cgcatggatc cccacagatg gtacggaggg
     5161 atatcgggct gtcggtgacg cacaggttct ccaccaagtc ctggctgtcg caggtctgcc
     5221 acgtgtgcca gaagagcatg atatttggag tgaagtgcaa gcattgcagg ttgaagtgtc
     5281 acaacaaatg taccaaagaa gcccctgcct gtagaatatc cttcctgcca ctaactcggc
     5341 tcgagctcaa gcttcgaatt ctgcagtcga cggtaccgcg ggcccgggat ccaccggtcg
     5401 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     5461 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     5521 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     5581 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     5641 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     5701 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     5761 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     5821 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     5881 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     5941 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     6001 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     6061 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     6121 agtaaggcct gtcaggccaa gcttggggat caattctcta gagctcgctg atcagcctcg
     6181 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     6241 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     6301 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     6361 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     6421 agaaccagct gggcctacta gatagcggac cccttaccga aacatcgccg cattctgcag
     6481 aggagtcgaa cttagtgtat gtaaacttct gacccactgg aattgtgata cagtgaatta
     6541 taagtgaaat aatctgtctg taaacaattg ttggaaaaat gacttgtgtc atgcacaaag
     6601 tagatgtcct aactgacttg ccaaaactat tgtttgttaa caagaaattt gtggagtagt
     6661 tgaaaaacga gttttaatga ctccaactta agtgtatgta aacttccgac ttcaactgta
     6721 tagggatcc
//
