LOCUS       sequence-435787-        6572 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for ceramide (for bacterial expression and 
            purification).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6572)
  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 6572)
  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: pETM11-SUMO3-KSR1-CA3-sfGFP (C-KSR)
FEATURES             Location/Qualifiers
     source          1..6572
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    43..67
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             82..104
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             117..134
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             165..185
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             729..1442
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             1460..1477
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1530..1548)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1544..1591
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1628..2083
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1715..1734)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1925..1946
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2175..2990)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2175..2990)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2898..2917
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2982..3001
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3112..3700
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3601..3620
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3854..3871
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3886..4025
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3951..3970)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4111..4133
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4127..4318)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5127..6209)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6171..6190
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6210..6287)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6493..6512
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
       61 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgaaacatc
      121 accatcacca tcaccccatg agcgattacg acatccccac tactgagaat ctttattttc
      181 agggcgccat gggcaacgat cacattaacc tgaaagtggc cggtcaagac ggtagcgtag
      241 tccagtttaa aatcaaacgc cacacccctc tgtcgaaact gatgaaagcc tattgtgaac
      301 gccaaggtct gtctatgcgt cagatccgtt ttcgcttcga tggacagccg attaacgaaa
      361 ccgacactcc agcacagctg gaaatggaag atgaggacac cattgacgtg ttccagcaac
      421 agaccggtgg atccaccatg gggaaccgca ttgatgacgt ctcctcgatg aggtttgatc
      481 tctcgcatgg atccccacag atggtacgga gggatatcgg gctgtcggtg acgcacaggt
      541 tctccaccaa gtcctggctg tcgcaggtct gccacgtgtg ccagaagagc atgatatttg
      601 gagtgaagtg caagcattgc aggttgaagt gtcacaacaa atgtaccaaa gaagcccctg
      661 cctgtagaat atccttcctg ccactaactc gggcaggtcc gggttctatt gcgactatgg
      721 tcgaattctc taaaggcgag gaactgttta cgggcgtcgt tccaatcctg gtagaactgg
      781 atggtgacgt gaacggccac aaattttccg tacgtggtga aggcgaaggc gatgcaacca
      841 acggtaaact gaccctcaaa tttatctgca ccactggcaa actgccggtt ccgtggccaa
      901 ccctggtgac caccctgact tacggtgtac agtgtttctc tcgttatccg gaccatatga
      961 aacgtcacga ctttttcaaa agcgcaatgc cggagggtta cgttcaggaa cgtacgattt
     1021 ctttcaaaga cgacggcacg tacaaaactc gcgctgaagt gaaattcgaa ggcgataccc
     1081 tggttaaccg catcgagctg aaaggcatcg atttcaaaga ggacggcaac atcctgggtc
     1141 acaaactgga atacaacttc aactctcata acgtgtatat caccgcggat aaacagaaaa
     1201 acggcatcaa agccaacttc aaaatccgtc acaacgtgga agacggttct gtacagctgg
     1261 cggaccacta tcagcagaac accccaattg gtgacggtcc ggttctgctg ccagataacc
     1321 attacctgag cactcagtcc gtgctgtcca aagatccgaa cgaaaaacgc gatcacatgg
     1381 tcctcctgga gttcgttact gccgctggta ttacccacgg tatggacgaa ctgtacaaat
     1441 gataagcggc cgcctcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
     1501 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     1561 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     1621 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1681 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     1741 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     1801 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     1861 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     1921 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     1981 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2041 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     2101 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2161 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     2221 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     2281 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     2341 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     2401 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     2461 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     2521 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     2581 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     2641 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     2701 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     2761 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     2821 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     2881 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     2941 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     3001 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga
     3061 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3121 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3181 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3241 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3301 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3361 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3421 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3481 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3541 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3601 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3661 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3721 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3781 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3841 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3901 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     3961 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     4021 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4081 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4141 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     4201 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     4261 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     4321 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     4381 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     4441 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     4501 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     4561 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     4621 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     4681 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     4741 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     4801 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4861 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4921 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4981 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     5041 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     5101 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5161 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     5221 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     5281 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     5341 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     5401 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     5461 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     5521 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     5581 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     5641 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     5701 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     5761 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     5821 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     5881 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     5941 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     6001 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     6061 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     6121 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     6181 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     6241 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     6301 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     6361 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     6421 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     6481 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     6541 gatgccggcc acgatgcgtc cggcgtagag ga
//
