LOCUS       sequence-481099-        5522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of syntaxin-1(I233A,K84TAG,R210H) fused to 
            mCherry for genetic code expansion via Amber codon supression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5522)
  AUTHORS   Obashi K, Strub MP, Taraska JW
  TITLE     A PIP2-stabilized syntaxin-1a structure mapped with transition metal
            ion FRET and unnatural fluorescent amino acids at the plasma 
            membrane.
  JOURNAL   Structure. 2025 Nov 24:S0969-2126(25)00388-0. doi: 
            10.1016/j.str.2025.10.001.
  PUBMED    41290003
REFERENCE   2  (bases 1 to 5522)
  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: "Structure. 
            2025 Nov 24:S0969-2126(25)00388-0. doi: 10.1016/j.str.2025.10.001."
COMMENT     Sequence Label: Syntaxin1A(I233A,R210H,K84TAG)-mCherry
FEATURES             Location/Qualifiers
     source          1..5522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1495..2202
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELY"
     primer_bind     complement(1640..1658)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1908..1927
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2353..2474
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2390..2409)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2444..2463
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2481..2935)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2618..2639)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2829..2848
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2962..3066
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        3068..3259
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3091..3111)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             3418..4212
                     /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             3418..4212
                     /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(3472..3491)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4082..4101
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4400..4419)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4444..4491
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4820..5408
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5309..5328
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatgaagga ccgaacccag gagctccgca cggccaagga cagcgatgac
      661 gacgatgatg tcactgtcac tgtggaccga gaccgcttca tggatgagtt ctttgaacag
      721 gtggaagaga tccggggctt tattgacaag attgctgaaa acgtggagga ggtgaagagg
      781 aaacacagcg ccatcctggc ctccccgaac cccgatgaga agaccaagga ggaactggag
      841 gagctcatgt cggacattaa gtagacagcg aacaaagttc gctccaagct aaagagcatc
      901 gagcagagca tcgagcagga ggaaggtctg aaccgctcgt cggcggacct gaggatccgg
      961 aagacgcagc attccacgct gtcccgaaag tttgtggagg tcatgtccga gtacaacgcc
     1021 actcagtcag actaccgaga acgctgcaaa gggcgcatcc agaggcagct ggagatcact
     1081 ggccggacca cgaccagtga ggagttggaa gacatgctgg agagtgggaa tcccgccatc
     1141 tttgcctctg ggatcatcat ggactccagc atctcgaagc aggccctcag tgagatcgag
     1201 accaggcaca gtgagatcat caagttggag aacagcatcc acgagctaca cgatatgttc
     1261 atggacatgg ccatgctggt ggagagccag ggggagatga ttgacagggc cgagtacaat
     1321 gtggaacacg ctgtggacta cgtggagagg gccgtgtctg acaccaagaa ggccgtcaag
     1381 taccagagca aggcacgcag gaagaagatc atgatcatca tttgctgtgt gattctgggc
     1441 atcatcatcg cctccaccat cgggggcatc tttggaggcg gatccggagg gtctatggtg
     1501 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
     1561 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     1621 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     1681 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     1741 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     1801 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     1861 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     1921 aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
     1981 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     2041 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     2101 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     2161 gagggccgcc actccaccgg cggcatggac gagctgtact gagtcgacgg taccgcgggc
     2221 ccgggatcca ccggatctag ataactgatc ataatcagcc ataccacatt tgtagaggtt
     2281 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     2341 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     2401 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     2461 atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     2521 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     2581 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     2641 attaaagaac gtggactcca acgtcaaagg gcgaaaaccg tctatcaggg cgatggccca
     2701 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     2761 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     2821 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     2881 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     2941 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3001 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3061 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3121 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3181 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3241 agtcagcaac catagtcccg cccctaactc cgccccatgg ctgactaatt ttttttattt
     3301 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     3361 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg
     3421 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3481 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     3541 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     3601 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     3661 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     3721 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     3781 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     3841 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     3901 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc
     3961 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4021 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4081 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4141 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     4201 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     4261 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4321 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4381 accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta
     4441 tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg
     4501 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
     4561 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
     4621 tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt
     4681 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4741 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4801 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4861 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4921 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     4981 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5041 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5101 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5161 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5221 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5281 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5341 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5401 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5461 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc
     5521 at
//
