LOCUS       sequence-435975-        5341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for ceramide (putative, mammalian expression).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5341)
  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 5341)
  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: pNES-PRKCZ-C20-EGFP (C-NES-PRKCZ-C20)
FEATURES             Location/Qualifiers
     source          1..5341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      597..606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    1221..1273
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      1281..1290
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1287..1289,1290..1292,1293..2006)
                     /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(1287..1289,1290..1292,1293..2003)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1332..1353)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1593..1612)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1940..1961
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2129..2250
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2166..2185)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2220..2239
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2257..2712)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2394..2415)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2606..2625
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2739..2843
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2845..3202
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2845..3036
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2868..2888)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3053..3188
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3237..4031
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3237..4031
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3291..3310)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3901..3920
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4219..4238)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4263..4310
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4639..5227
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5128..5147
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcca
      601 ccatggctct gcagaaaaag ttggaagagc ttgagctgga tgaggcaccg gtcgccaccg
      661 gtgacacaac gagcactttc tgcggaaccc cgaattacat cgcccccgaa atcctgcggg
      721 gagaggagta cgggttcagc gtggactggt gggcgctggg agtcctcatg tttgagatga
      781 tggccgggcg ctccccgttc gacatcatca ccgacaaccc ggacatgaac acagaggact
      841 accttttcca agtgatcctg gagaagccca tccggatccc ccggttcctg tccgtcaaag
      901 cctcccatgt tttaaaagga tttttaaata aggaccccaa agagaggctc ggctgccggc
      961 cacagactgg attttctgac atcaagtccc acgcgttctt ccgcagcata gactgggact
     1021 tgctggagaa gaagcaggcg ctccctccat tccagccaca gatcacagac gactacggtc
     1081 tggacaactt tgacacacag ttcaccagcg agcccgtgca gctgacccca gacgatgagg
     1141 atgccataaa gaggatcgac cagtcagagt tcgaaggctt tgagtatatc aacccattat
     1201 tgctgtccac cgaggagtcg ctcgagctca agcttcgaat tctgcagtcg acggtaccgc
     1261 gggcccggga tccaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     1321 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1381 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1441 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1501 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1561 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1621 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1681 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     1741 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     1801 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1861 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     1921 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1981 tcggcatgga cgagctgtac aagtaaagcg gccgcgactc tagatcataa tcagccatac
     2041 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     2101 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     2161 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     2221 tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt aatattttgt
     2281 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     2341 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     2401 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     2461 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     2521 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     2581 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     2641 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     2701 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2761 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2821 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca
     2881 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2941 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3001 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3061 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3121 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     3181 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     3241 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3301 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3361 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     3421 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3481 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3541 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3601 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3661 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3721 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     3781 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3841 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3901 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3961 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4021 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     4081 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4141 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4201 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     4261 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     4321 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     4381 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     4441 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     4501 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4561 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4621 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4681 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4741 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     4801 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4861 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4921 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4981 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5041 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5101 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5161 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5221 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5281 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca
     5341 t
//
