LOCUS       sequence-348286-        7047 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Synchronize trafficking of Stx5LdeltaER (M55V) from the ER, for use 
            with FLIM (RUSH system).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7047)
  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 7047)
  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: str-KDEL_Stx5LdeltaER-SBP-mCitrine
FEATURES             Location/Qualifiers
     source          1..7047
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          1412..1641
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1624..1643
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(1713..2287,2288..2290,2291..2299)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1880..1897)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2107..2126
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3412..3522
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             join(3535..3537,3538..4251)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3838..3857)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    4380..4501
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4417..4436)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4656..4672)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4656..4672)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4669..4691)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4680..4696
                     /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)."
     promoter        complement(join(4704..4710,4711..4728,4729..4734))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4749..4770
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4887..4904)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5058..5646)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5138..5157)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5817..6608,6609..6677))
                     /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     6440..6459
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6678..6782)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6857..6876)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatcgat atctgcggcc tagctagcgc ttaaggcctg ttaaccggta
      721 tggatgtatg cgtccgtctt gccctgtggc tcctctgggg actcctcctg caccagggcc
      781 agagcctcag ccatagtcac agtgagaagg cgacaggaac cagctcgggc gccggtttaa
      841 acgaccctag caaagactca aaagctcagg tgtccgctgc cgaggctggc attactggaa
      901 catggtacaa tcagctcggg agcaccttta ttgtgactgc tggagccgat ggagccctca
      961 ccggaacata cgaatctgct gtgggaaacg ccgaatcacg gtacgtcctc actggccgat
     1021 acgatagtgc ccctgccacc gacggatctg ggactgccct gggatggact gtcgcttgga
     1081 aaaacaacta ccggaatgct cattctgcca caacatggag tggacagtac gtgggaggcg
     1141 ctgaggctag aatcaataca cagtggctgc tcacatctgg cacaaccgag gcaaatgctt
     1201 ggaaatccac cctggtggga catgacacat tcaccaaagt gaaaccctcc gccgcttcaa
     1261 tcgatgccgc caaaaaagcc ggagtcaaca acggcaatcc tctggatgcc gtccagcagc
     1321 tcggaccgaa agatgaactg tgagcggccg catagataac tgatccagtg tgctggaatt
     1381 aattcgctgt ctgcgagggc cagctgttgg ggtgagtact ccctctcaaa agcgggcatg
     1441 acttctgcgc taagattgtc agtttccaaa aacgaggagg atttgatatt cacctggccc
     1501 gcggtgatgc ctttgagggt ggccgcgtcc atctggtcag aaaagacaat ctttttgttg
     1561 tcaagcttga ggtgtggcag gcttgagatc tggccataca cttgagtgac aatgacatcc
     1621 actttgcctt tctctccaca ggtgtccact cccaggtcca actgcaggtc gagcatgcat
     1681 ctagggcggc caattccgcc cctctccccc cccccctctc cctccccccc ccctaacgtt
     1741 actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc
     1801 atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc
     1861 attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag
     1921 gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg
     1981 cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat
     2041 acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga
     2101 gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc
     2161 cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg
     2221 ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga
     2281 tgataagctt gccacaaccc gggaggcgcg cctaccatga ttcctgcagc tgcatacggg
     2341 tctaagaaca cggatcaggg tgtctacctg ggtctctcaa agacacaggt cctgtcccct
     2401 gcaactgctg gcagtagcag cagcgacatc gcccctctgc cccccccagt gaccctcgtc
     2461 cctccccctc ccgacaccgt gtcctgccgg gatcggaccc aggagtttct gtctgcctgc
     2521 aagtcgctgc agacccgtca gaatggaatc cagacaaata agccagcttt gcgtgctgtc
     2581 cgacaacgca gtgagttcac cctcatggcc aagcgcattg ggaaagacct tagcaacaca
     2641 tttgccaagc tggagaagct gacaatcttg gcaaagcgca agtccctctt tgatgataaa
     2701 gcagtggaaa ttgaagagct aacatatatc atcaaacagg acatcaatag cctcaacaaa
     2761 caaattgctc agctccagga tttcgtgaga gccaagggca gccagagtgg ccggcacctg
     2821 cagacccact ccaacaccat tgtggtctcc ttgcagtcga aactggcttc tatgtccaat
     2881 gacttcaaat cggttttaga agtgaggaca gagaacctga agcagcagag gagccggaga
     2941 gagcagttct cccgggcacc tgtgtcagcc ctgccccttg cccctaacca cctgggcggt
     3001 ggtgctgtgg ttctgggggc agagtcccat gcctccaagg atgtcgccat cgacatgatg
     3061 gactctcgga ccagccagca gctgcagctc attgacgagc aggattccta catccagagt
     3121 cgggcagaca ccatgcagaa cattgagtcg acaattgttg agttgggctc catctttcag
     3181 cagttggcac acatggttaa ggaacaggag gaaaccattc agaggatcga cgagaacgtg
     3241 ctaggagccc agctggacgt tgaggccgcc cattcagaga tcctcaagta cttccagtct
     3301 gtcacctcca accggtggct catggtcaaa atcttcctca tcctcattgt cttcttcatc
     3361 atctttgtgg tcttccttgc tcggggcggc agcggcggca gcacgaattc cgacgagaag
     3421 accactggtt ggcgaggtgg acacgttgtt gaaggactgg ctggggaact tgaacaactt
     3481 cgtgcacgac tggagcatca cccacaaggt caacgtgaac cacctgcagg tgctgtgagc
     3541 aagggcgagg agctgttcac cggcgtggtg cccatcctgg tggagctgga cggcgacgtg
     3601 aacggccaca agttcagcgt gagcggcgag ggcgagggcg acgccaccta cggcaagctg
     3661 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctggtgacc
     3721 accttcggct acggcctgat gtgcttcgcc cgctaccccg accacatgaa gcagcacgac
     3781 ttcttcaaga gcgccatgcc cgagggctac gtgcaggagc gcaccatctt cttcaaggac
     3841 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     3901 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctgggcca caagctggag
     3961 tacaactaca acagccacaa cgtgtacatc atggccgaca agcagaagaa cggcatcaag
     4021 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctggc cgaccactac
     4081 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     4141 taccagagca agctgagcaa ggaccccaac gagaagcgcg accacatggt gctgctggag
     4201 ttcgtgaccg ccgccggcat caccctgggc atggacgagc tgtacaagta agcggccgcg
     4261 actctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     4321 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     4381 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4441 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4501 aacgcgtcga gtgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt
     4561 cgagtgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     4621 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4681 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4741 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4801 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4861 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4921 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4981 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5041 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5101 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5161 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5221 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5281 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5341 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5401 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5461 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5521 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5581 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5641 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5701 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5761 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5821 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5881 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5941 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6001 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6061 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6121 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6181 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6241 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6301 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6361 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6421 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6481 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6541 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6601 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6661 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6721 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6781 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     6841 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     6901 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
     6961 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
     7021 gcgatgtacg ggccagatat acgcgtt
//
