LOCUS       sequence-435788-        6740 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for glucosyl-ceramide (putative, mammalian 
            expression).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6740)
  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 6740)
  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- sfGFP-KSR1-CA3  (N-KSR)
FEATURES             Location/Qualifiers
     source          1..6740
                     /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             435..452
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             510..1220
                     /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             1518..1541
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1578..1601
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1628..1645
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1698..1716)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1712..1759
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1796..2251
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1883..1902)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2093..2114
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2343..3158)
                     /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(2343..3158)
                     /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     3066..3085
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3150..3169
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3280..3868
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3769..3788
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4022..4039
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4054..4193
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4119..4138)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4279..4301
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4295..4486)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5295..6377)
                     /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     6339..6358
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6378..6455)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6661..6680
                     /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 atccctggtt ccacgtggta gcaactccgc tgttgatggt accgcaggtc
      481 cgggttctat tgcgactatg gtcgaattct ctaaaggcga ggaactgttt acgggcgtcg
      541 ttccaatcct ggtagaactg gatggtgacg tgaacggcca caaattttcc gtacgtggtg
      601 aaggcgaagg cgatgcaacc aacggtaaac tgaccctcaa atttatctgc accactggca
      661 aactgccggt tccgtggcca accctggtga ccaccctgac ttacggtgta cagtgtttct
      721 ctcgttatcc ggaccatatg aaacgtcacg actttttcaa aagcgcaatg ccggagggtt
      781 acgttcagga acgtacgatt tctttcaaag acgacggcac gtacaaaact cgcgctgaag
      841 tgaaattcga aggcgatacc ctggttaacc gcatcgagct gaaaggcatc gatttcaaag
      901 aggacggcaa catcctgggt cacaaactgg aatacaactt caactctcat aacgtgtata
      961 tcaccgcgga taaacagaaa aacggcatca aagccaactt caaaatccgt cacaacgtgg
     1021 aagacggttc tgtacagctg gcggaccact atcagcagaa caccccaatt ggtgacggtc
     1081 cggttctgct gccagataac cattacctga gcactcagtc cgtgctgtcc aaagatccga
     1141 acgaaaaacg cgatcacatg gtcctcctgg agttcgttac tgccgctggt attacccacg
     1201 gtatggacga actgtacaaa ggatccggcc cacagggcct ggggaaccgc attgatgacg
     1261 tctcctcgat gaggtttgat ctctcgcatg gatccccaca gatggtacgg agggatatcg
     1321 ggctgtcggt gacgcacagg ttctccacca agtcctggct gtcgcaggtc tgccacgtgt
     1381 gccagaagag catgatattt ggagtgaagt gcaagcattg caggttgaag tgtcacaaca
     1441 aatgtaccaa agaagcccct gcctgtagaa tatcattcct gccactaact cggtaaggcc
     1501 atatcggcct taagctttgg agccacccgc agttcgagaa aggtggaggt tccggaggtg
     1561 gatcgggagg tggatcgtgg agccacccgc agttcgaaaa agcggccgat taagcggccg
     1621 cctcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     1681 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     1741 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     1801 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     1861 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     1921 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     1981 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2041 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2101 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2161 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2221 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     2281 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     2341 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     2401 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     2461 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     2521 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     2581 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     2641 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     2701 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     2761 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     2821 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     2881 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     2941 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     3001 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     3061 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     3121 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     3181 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     3241 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3301 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3361 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3421 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3481 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3541 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3601 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3661 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3721 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3781 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3841 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3901 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3961 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4021 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     4081 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     4141 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     4201 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4261 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4321 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     4381 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     4441 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4501 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4561 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4621 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     4681 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     4741 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4801 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     4861 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4921 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     4981 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     5041 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     5101 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     5161 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     5221 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     5281 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5341 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     5401 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     5461 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     5521 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     5581 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     5641 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     5701 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     5761 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     5821 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     5881 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     5941 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     6001 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     6061 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     6121 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     6181 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     6241 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     6301 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     6361 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     6421 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     6481 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     6541 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     6601 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     6661 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     6721 gatgcgtccg gcgtagagga
//
