LOCUS       sequence-413647-        6991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Synchronize trafficking of Stx5L (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 6991)
  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 6991)
  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_Stx5L(M55V)-SBP-mCitrine
FEATURES             Location/Qualifiers
     source          1..6991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          1646..1875
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1858..1877
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(1947..2521,2522..2524,2525..2533)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2114..2131)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2341..2360
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3643..3753
                     /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(3766..3768,3769..4482)
                     /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(4069..4088)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    4611..4732
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4648..4667)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4834..4850)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4834..4850)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4847..4869)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4858..4874
                     /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(4882..4888,4889..4906,4907..4912))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4927..4948
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5065..5082)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5236..5824)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5316..5335)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5995..6786,6787..6855))
                     /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     6618..6637
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6856..6960)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat cgatatctgc ggcctagcta gcgcttaagg cctgttaacc ggtatggatg
      961 tatgcgtccg tcttgccctg tggctcctct ggggactcct cctgcaccag ggccagagcc
     1021 tcagccatag tcacagtgag aaggcgacag gaaccagctc gggcgccggt ttaaacgacc
     1081 ctagcaaaga ctcaaaagct caggtgtccg ctgccgaggc tggcattact ggaacatggt
     1141 acaatcagct cgggagcacc tttattgtga ctgctggagc cgatggagcc ctcaccggaa
     1201 catacgaatc tgctgtggga aacgccgaat cacggtacgt cctcactggc cgatacgata
     1261 gtgcccctgc caccgacgga tctgggactg ccctgggatg gactgtcgct tggaaaaaca
     1321 actaccggaa tgctcattct gccacaacat ggagtggaca gtacgtggga ggcgctgagg
     1381 ctagaatcaa tacacagtgg ctgctcacat ctggcacaac cgaggcaaat gcttggaaat
     1441 ccaccctggt gggacatgac acattcacca aagtgaaacc ctccgccgct tcaatcgatg
     1501 ccgccaaaaa agccggagtc aacaacggca atcctctgga tgccgtccag cagctcggac
     1561 cgaaagatga actgtgagcg gccgcataga taactgatcc agtgtgctgg aattaattcg
     1621 ctgtctgcga gggccagctg ttggggtgag tactccctct caaaagcggg catgacttct
     1681 gcgctaagat tgtcagtttc caaaaacgag gaggatttga tattcacctg gcccgcggtg
     1741 atgcctttga gggtggccgc gtccatctgg tcagaaaaga caatcttttt gttgtcaagc
     1801 ttgaggtgtg gcaggcttga gatctggcca tacacttgag tgacaatgac atccactttg
     1861 cctttctctc cacaggtgtc cactcccagg tccaactgca ggtcgagcat gcatctaggg
     1921 cggccaattc cgcccctctc cccccccccc tctccctccc ccccccctaa cgttactggc
     1981 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg
     2041 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct
     2101 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca
     2161 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg
     2221 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct
     2281 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa
     2341 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt
     2401 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa
     2461 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa
     2521 gcttgccaca acccgggagg cgcgccaatg atcccgcgga aacgctacgg gtctaagaac
     2581 acggatcagg gtgtctacct gggtctctca aagacacagg tcctgtcccc tgcaactgct
     2641 ggcagtagca gcagcgacat cgcccctctg ccccccccag tgaccctcgt ccctccccct
     2701 cccgacaccg tgtcctgccg ggatcggacc caggagtttc tgtctgcctg caagtcgctg
     2761 cagacccgtc agaatggaat ccagacaaat aagccagctt tgcgtgctgt ccgacaacgc
     2821 agtgagttca ccctcatggc caagcgcatt gggaaagacc ttagcaacac atttgccaag
     2881 ctggagaagc tgacaatctt ggcaaagcgc aagtccctct ttgatgataa agcagtggaa
     2941 attgaagagc taacatatat catcaaacag gacatcaata gcctcaacaa acaaattgct
     3001 cagctccagg atttcgtgag agccaagggc agccagagtg gccggcacct gcagacccac
     3061 tccaacacca ttgtggtctc cttgcagtcg aaactggctt ctatgtccaa tgacttcaaa
     3121 tcggttttag aagtgaggac agagaacctg aagcagcaga ggagccggag agagcagttc
     3181 tcccgggcac ctgtgtcagc cctgcccctt gcccctaacc acctgggcgg tggtgctgtg
     3241 gttctggggg cagagtccca tgcctccaag gatgtcgcca tcgacatgat ggactctcgg
     3301 accagccagc agctgcagct cattgacgag caggattcct acatccagag tcgggcagac
     3361 accatgcaga acattgagtc gacaattgtt gagttgggct ccatctttca gcagttggca
     3421 cacatggtta aggaacagga ggaaaccatt cagaggatcg acgagaacgt gctaggagcc
     3481 cagctggacg ttgaggccgc ccattcagag atcctcaagt acttccagtc tgtcacctcc
     3541 aaccggtggc tcatggtcaa aatcttcctc atcctcattg tcttcttcat catctttgtg
     3601 gtcttccttg ctcggggcgg cagcggcggc agcacgaatt ccgacgagaa gaccactggt
     3661 tggcgaggtg gacacgttgt tgaaggactg gctggggaac ttgaacaact tcgtgcacga
     3721 ctggagcatc acccacaagg tcaacgtgaa ccacctgcag gtgctgtgag caagggcgag
     3781 gagctgttca ccggcgtggt gcccatcctg gtggagctgg acggcgacgt gaacggccac
     3841 aagttcagcg tgagcggcga gggcgagggc gacgccacct acggcaagct gaccctgaag
     3901 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctggtgac caccttcggc
     3961 tacggcctga tgtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
     4021 agcgccatgc ccgagggcta cgtgcaggag cgcaccatct tcttcaagga cgacggcaac
     4081 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     4141 aagggcatcg acttcaagga ggacggcaac atcctgggcc acaagctgga gtacaactac
     4201 aacagccaca acgtgtacat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     4261 aagatccgcc acaacatcga ggacggcagc gtgcagctgg ccgaccacta ccagcagaac
     4321 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagagc
     4381 aagctgagca aggaccccaa cgagaagcgc gaccacatgg tgctgctgga gttcgtgacc
     4441 gccgccggca tcaccctggg catggacgag ctgtacaagt aagcggccgc gactctagat
     4501 aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     4561 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     4621 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4681 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtcg
     4741 agtgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     4801 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4861 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     4921 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4981 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5041 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5101 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5161 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5221 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5281 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5341 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5401 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5461 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5521 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5581 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5641 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5701 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5761 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5821 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5881 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5941 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6001 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6061 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6121 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6181 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6241 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6301 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6361 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6421 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6481 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6541 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6601 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6661 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6721 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6781 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6841 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6901 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6961 cacatttccc cgaaaagtgc cacctgacgt c
//
