LOCUS       sequence-413654-        5787 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Stx5 M55V mutant fused to GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5787)
  AUTHORS   Linders PTA, Gerretsen ECF, Ashikov A, Vals MA, de Boer R, Revelo 
            NH, Arts R, Baerenfaenger M, Zijlstra F, Huijben K, Raymond K, Muru 
            K, Fjodorova O, Pajusalu S, Ounap K, Ter Beest M, Lefeber D, van den
            Bogaart G
  TITLE     Congenital disorder of glycosylation caused by starting 
            site-specific variant in syntaxin-5.
  JOURNAL   Nat Commun. 2021 Oct 28;12(1):6227. doi: 10.1038/s41467-021-26534-y.
  PUBMED    34711829
REFERENCE   2  (bases 1 to 5787)
  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: "Nat 
            Commun."; date: "2021-10-28"; volume: "12(1):6227. doi"; pages: " 
            10.1038/s41467-021-26534-y"
COMMENT     Sequence Label: Stx5L(m55V)-GFP
FEATURES             Location/Qualifiers
     source          1..5787
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        63..366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        367..570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     521..541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(1738..1740,1741..2454)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1738..1740,1741..2451)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1780..1801)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2041..2060)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2388..2409
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2577..2698
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2614..2633)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2668..2687
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2705..3160)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2842..2863)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3054..3073
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3187..3291
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3293..3650
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3293..3484
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3316..3336)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3501..3636
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3685..4479
                     /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             3685..4479
                     /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(3739..3758)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4349..4368
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4667..4686)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4711..4758
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5087..5675
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5576..5595
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      421 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      481 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      541 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
      601 taccggactc agatctcgag ctcaagcttc gaattcatga tcccccgcaa gcgctacggc
      661 agcaagaaca ccgaccaggg cgtgtacctg ggcctgagca agacccaggt gctgagcccc
      721 gccaccgccg gcagcagcag cagcgacatc gcccccctgc ccccccccgt gaccctggtg
      781 cccccccccc ccgacaccgt gagctgccgc gaccgcaccc aggagttcct gagcgcctgc
      841 aagagcctgc agacccgcca gaacggcatc cagaccaaca agcccgccct gcgcgccgtg
      901 cgccagcgca gcgagttcac cctgatggcc aagcgcatcg gcaaggacct gagcaacacc
      961 ttcgccaagc tggagaagct gaccatcctg gccaagcgca agagcctgtt cgacgacaag
     1021 gccgtggaga tcgaggagct gacctacatc atcaagcagg acatcaacag cctgaacaag
     1081 cagatcgccc agctgcagga cttcgtgcgc gccaagggca gccagagcgg ccgccacctg
     1141 cagacccaca gcaacaccat cgtggtgagc ctgcagagca agctggccag catgagcaac
     1201 gacttcaaga gcgtgctgga ggtgcgcacc gagaacctga agcagcagcg cagccgccgc
     1261 gagcagttca gccgcgcccc cgtgagcgcc ctgcccctgg cccccaacca cctgggcggc
     1321 ggcgccgtgg tgctgggcgc cgagagccac gccagcaagg acgtggccat cgacatgatg
     1381 gacagccgca ccagccagca gctgcagctg atcgacgagc aggacagcta catccagagc
     1441 cgcgccgaca ccatgcagaa catcgagagc accatcgtgg agctgggcag catcttccag
     1501 cagctggccc acatggtgaa ggagcaggag gagaccatcc agcgcatcga cgagaacgtg
     1561 ctgggcgccc agctggacgt ggaggccgcc cacagcgaga tcctgaagta cttccagagc
     1621 gtgaccagca accgctggct gatggtgaag atcttcctga tcctgatcgt gttcttcatc
     1681 atcttcgtgg tgttcctggc ccgggatcca ccggtaggag gaagcggagg aagcgctgtg
     1741 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1801 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1861 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1921 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     1981 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     2041 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     2101 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     2161 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     2221 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     2281 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     2341 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     2401 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc
     2461 cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
     2521 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     2581 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     2641 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag
     2701 gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     2761 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     2821 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     2881 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     2941 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     3001 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     3061 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     3121 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga
     3181 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3241 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg
     3301 gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     3361 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     3421 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     3481 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3541 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     3601 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa
     3661 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3721 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3781 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3841 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg
     3901 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3961 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4021 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4081 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4141 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4201 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4261 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4321 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4381 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4441 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     4501 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     4561 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4621 gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg
     4681 agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg
     4741 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata
     4801 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc
     4861 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca
     4921 tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4981 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     5041 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5101 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5161 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5221 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5281 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5341 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5401 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5461 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5521 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5581 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5641 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5701 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     5761 ctgtggataa ccgtattacc gccatgc
//
