LOCUS       sequence-325019-        6827 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Synchronize trafficking of Stx5S 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 6827)
  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 6827)
  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_Stx5S-SBP-mCitrine
FEATURES             Location/Qualifiers
     source          1..6827
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..75)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        247..626
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        627..830
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     781..801
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     875..894
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        875..893
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          1658..1887
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1870..1889
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(1959..2533,2534..2536,2537..2545)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2126..2143)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2353..2372
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3491..3601
                     /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(3614..3616,3617..4330)
                     /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(3917..3936)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    4459..4580
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4496..4515)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4682..4698)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4682..4698)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4695..4717)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4706..4722
                     /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(4730..4736,4737..4754,4755..4760))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4775..4796
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4913..4930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5084..5672)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5164..5183)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5843..6634,6635..6703))
                     /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     6466..6485
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6704..6808)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
       61 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      121 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      181 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      241 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      301 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      361 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      421 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      481 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      541 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      601 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      661 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      721 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      781 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
      841 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
      901 aagcttggta ccgagctcgg atcgatatct gcggcctagc tagcgcttaa ggcctgttaa
      961 ccggtatgga tgtatgcgtc cgtcttgccc tgtggctcct ctggggactc ctcctgcacc
     1021 agggccagag cctcagccat agtcacagtg agaaggcgac aggaaccagc tcgggcgccg
     1081 gtttaaacga ccctagcaaa gactcaaaag ctcaggtgtc cgctgccgag gctggcatta
     1141 ctggaacatg gtacaatcag ctcgggagca cctttattgt gactgctgga gccgatggag
     1201 ccctcaccgg aacatacgaa tctgctgtgg gaaacgccga atcacggtac gtcctcactg
     1261 gccgatacga tagtgcccct gccaccgacg gatctgggac tgccctggga tggactgtcg
     1321 cttggaaaaa caactaccgg aatgctcatt ctgccacaac atggagtgga cagtacgtgg
     1381 gaggcgctga ggctagaatc aatacacagt ggctgctcac atctggcaca accgaggcaa
     1441 atgcttggaa atccaccctg gtgggacatg acacattcac caaagtgaaa ccctccgccg
     1501 cttcaatcga tgccgccaaa aaagccggag tcaacaacgg caatcctctg gatgccgtcc
     1561 agcagctcgg accgaaagat gaactgtgag cggccgcata gataactgat ccagtgtgct
     1621 ggaattaatt cgctgtctgc gagggccagc tgttggggtg agtactccct ctcaaaagcg
     1681 ggcatgactt ctgcgctaag attgtcagtt tccaaaaacg aggaggattt gatattcacc
     1741 tggcccgcgg tgatgccttt gagggtggcc gcgtccatct ggtcagaaaa gacaatcttt
     1801 ttgttgtcaa gcttgaggtg tggcaggctt gagatctggc catacacttg agtgacaatg
     1861 acatccactt tgcctttctc tccacaggtg tccactccca ggtccaactg caggtcgagc
     1921 atgcatctag ggcggccaat tccgcccctc tccccccccc cctctccctc ccccccccct
     1981 aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt
     2041 tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg
     2101 acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc
     2161 gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt
     2221 tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta
     2281 taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg
     2341 gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag
     2401 gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag
     2461 tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa
     2521 acacgatgat aagcttgcca caacccggga ggcgcgcctg agctgccgcg accgcaccca
     2581 ggagttcctg agcgcctgca agagcctgca gacccgccag aacggcatcc agaccaacaa
     2641 gcccgccctg cgcgccgtgc gccagcgcag cgagttcacc ctgatggcca agcgcatcgg
     2701 caaggacctg agcaacacct tcgccaagct ggagaagctg accatcctgg ccaagcgcaa
     2761 gagcctgttc gacgacaagg ccgtggagat cgaggagctg acctacatca tcaagcagga
     2821 catcaacagc ctgaacaagc agatcgccca gctgcaggac ttcgtgcgcg ccaagggcag
     2881 ccagagcggc cgccacctgc agacccacag caacaccatc gtggtgagcc tgcagagcaa
     2941 gctggccagc atgagcaacg acttcaagag cgtgctggag gtgcgcaccg agaacctgaa
     3001 gcagcagcgc agccgccgcg agcagttcag ccgcgccccc gtgagcgccc tgcccctggc
     3061 ccccaaccac ctgggcggcg gcgccgtggt gctgggcgcc gagagccacg ccagcaagga
     3121 cgtggccatc gacatgatgg acagccgcac cagccagcag ctgcagctga tcgacgagca
     3181 ggacagctac atccagagcc gcgccgacac catgcagaac atcgagagca ccatcgtgga
     3241 gctgggcagc atcttccagc agctggccca catggtgaag gagcaggagg agaccatcca
     3301 gcgcatcgac gagaacgtgc tgggcgccca gctggacgtg gaggccgccc acagcgagat
     3361 cctgaagtac ttccagagcg tgaccagcaa ccgctggctg atggtgaaga tcttcctgat
     3421 cctgatcgtg ttcttcatca tcttcgtggt gttcctggcc cgggatccac cggtcgccac
     3481 cttgaattcc gacgagaaga ccactggttg gcgaggtgga cacgttgttg aaggactggc
     3541 tggggaactt gaacaacttc gtgcacgact ggagcatcac ccacaaggtc aacgtgaacc
     3601 acctgcaggt gctgtgagca agggcgagga gctgttcacc ggcgtggtgc ccatcctggt
     3661 ggagctggac ggcgacgtga acggccacaa gttcagcgtg agcggcgagg gcgagggcga
     3721 cgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     3781 ctggcccacc ctggtgacca ccttcggcta cggcctgatg tgcttcgccc gctaccccga
     3841 ccacatgaag cagcacgact tcttcaagag cgccatgccc gagggctacg tgcaggagcg
     3901 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     3961 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     4021 cctgggccac aagctggagt acaactacaa cagccacaac gtgtacatca tggccgacaa
     4081 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     4141 gcagctggcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     4201 cgacaaccac tacctgagct accagagcaa gctgagcaag gaccccaacg agaagcgcga
     4261 ccacatggtg ctgctggagt tcgtgaccgc cgccggcatc accctgggca tggacgagct
     4321 gtacaagtaa gcggccgcga ctctagataa ctgatcataa tcagccatac cacatttgta
     4381 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     4441 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4501 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4561 aaactcatca atgtatctta acgcgtcgag tgcattctag ttgtggtttg tccaaactca
     4621 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4681 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4741 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4801 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4861 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4921 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4981 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5041 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5101 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5161 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5221 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5281 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5341 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5401 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5461 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5521 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5581 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5641 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5701 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5761 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5821 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5881 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5941 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6001 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6061 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6121 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6181 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6241 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6301 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6361 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6421 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6481 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6541 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6601 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6661 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6721 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6781 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtg
//
