LOCUS       sequence-369208-        6859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Stim1 mutant D76A fused to YFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6859)
  AUTHORS   Zamponi E, Meehl JB, Voeltz GK
  TITLE     The ER ladder is a unique morphological feature of developing 
            mammalian axons.
  JOURNAL   Dev Cell. 2022 Jun 6;57(11):1369-1382.e6. doi: 
            10.1016/j.devcel.2022.05.002. Epub 2022 May 23.
  PUBMED    35609616
REFERENCE   2  (bases 1 to 6859)
  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"; doi: 
            "10.1016/j.devcel.2022.05.002"; journalName: "Dev Cell"; date: 
            "2022-06-6- 6"; volume: "57"; issue: "11"; pages: "1369-1382.e6"
COMMENT     Sequence Label: YFP-Stim1-D76A
FEATURES             Location/Qualifiers
     source          1..6859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      618..627
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(624..626,627..629,630..1340)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(669..690)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(930..949)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1277..1298
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1350..1374
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3370..3394)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3398..3463
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    3587..3708
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3624..3643)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3678..3697
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3715..4170)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3852..3873)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4064..4083
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4197..4301
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4303..4660
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4303..4494
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4326..4346)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4511..4646
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4695..5489
                     /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             4695..5489
                     /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(4749..4768)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5359..5378
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5677..5696)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5721..5768
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6097..6685
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6586..6605
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcacca
      541 tggatgtatg cgtccgtctt gccctgtggc tcctctgggg actcctcctg caccagggcc
      601 agagcctcgc accggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg
      661 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
      721 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
      781 agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg cagtgcttcg
      841 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
      901 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
      961 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1021 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     1081 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     1141 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     1201 ccgtgctgct gcccgacaac cactacctga gctaccagtc cgccctgagc aaagacccca
     1261 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     1321 gcatggacga gctgtacaag tccggatcaa caagtttgta caaaaaagca ggctccctca
     1381 gccatagtca cagtgagaag gcgacaggaa ccagctcggg ggccaactct gaggagtcca
     1441 ctgcagcaga gttttgccga attgacaagc ccctgtgtca cagtgaggat gagaaactca
     1501 gcttcgaggc agtccgtaac atccacaaac tgatggccga tgatgccaat ggtgatgtgg
     1561 atgtggaaga aagtgatgag ttcctgaggg aagacctcaa ttaccatgac ccaacagtga
     1621 aacacagcac cttccatggt gaggataagc tcatcagcgt ggaggacctg tggaaggcat
     1681 ggaagtcatc agaagtatac aattggaccg tggatgaggt ggtacagtgg ctgatcacat
     1741 atgtggagct gcctcagtat gaggagacct tccggaagct gcagctcagt ggccatgcca
     1801 tgccaaggct ggctgtcacc aacaccacca tgacagggac tgtgctgaag atgacagacc
     1861 ggagtcatcg gcagaagctg cagctgaagg ctctggatac agtgctcttt gggcctcctc
     1921 tcttgactcg ccataatcac ctcaaggact tcatgctggt ggtgtctatc gttattggtg
     1981 tgggcggctg ctggtttgcc tatatccaga accgttactc caaggagcac atgaagaaga
     2041 tgatgaagga cttggagggg ttacaccgag ctgagcagag tctgcatgac cttcaggaaa
     2101 ggctgcacaa ggcccaggag gagcaccgca cagtggaggt ggagaaggtc catctggaaa
     2161 agaagctgcg cgatgagatc aaccttgcta agcaggaagc ccagcggctg aaggagctgc
     2221 gggagggtac tgagaatgag cggagccgcc aaaaatatgc tgaggaggag ttggagcagg
     2281 ttcgggaggc cttgaggaaa gcagagaagg agctagaatc tcacagctca tggtatgctc
     2341 cagaggccct tcagaagtgg ctgcagctga cacatgaggt ggaggtgcaa tattacaaca
     2401 tcaagaagca aaatgctgag aagcagctgc tggtggccaa ggagggggct gagaagataa
     2461 aaaagaagag aaacacactc tttggcacct tccacgtggc ccacagctct tccctggatg
     2521 atgtagatca taaaattcta acagctaagc aagcactgag cgaggcgaca gcagcattgc
     2581 gggagcgcct gcaccgctgg caacagatcg agatcctctg tggcttccag attgtcaaca
     2641 accctggcat ccactcactg gtggctgccc tcaacataga ccccagctgg atgggcagta
     2701 cacgccccaa ccctgctcac ttcatcatga ctgacgacgt ggatgacatg gatgaggaga
     2761 ttgtgtctcc cttgtccatg cagtccccta gcctgcagag cagtgttcgg cagcgcctga
     2821 cggagccaca gcatggcctg ggatctcaga gggatttgac ccattccgat tcggagtcct
     2881 ccctccacat gagtgaccgc cagcgtgtgg cccccaaacc tcctcagatg agccgtgctg
     2941 cagacgaggc tctcaatgcc atgacttcca atggcagcca ccggctgatc gagggggtcc
     3001 acccagggtc tctggtggag aaactgcctg acagccctgc cctggccaag aaggcattac
     3061 tggcgctgaa ccatgggctg gacaaggccc acagcctgat ggagctgagc ccctcagccc
     3121 cacctggtgg ctctccacat ttggattctt cccgttctca cagccccagc tccccagacc
     3181 cagacacacc atctccagtt ggggacagcc gagccctgca agccagccga aacacacgca
     3241 ttccccacct ggctggcaag aaggctgtgg ctgaggagga taatggctct attggcgagg
     3301 aaacagactc cagcccaggc cggaagaagt ttcccctcaa aatctttaag aagcctctta
     3361 agaagtagaa cccagctttc ttgtacaaag tggttgatcc ggactcagat ctcgagctca
     3421 agcttcgaat tctgcagtcg acggtaccgc gggcccggga tccaccggat ctagataact
     3481 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     3541 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     3601 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     3661 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtaaattg
     3721 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     3781 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     3841 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     3901 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     3961 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     4021 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     4081 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     4141 ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg
     4201 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4261 aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca
     4321 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     4381 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     4441 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     4501 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4561 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     4621 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
     4681 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     4741 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     4801 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     4861 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     4921 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     4981 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     5041 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     5101 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     5161 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     5221 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     5281 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     5341 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     5401 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     5461 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     5521 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     5581 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     5641 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     5701 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     5761 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     5821 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     5881 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     5941 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6001 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6061 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6121 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6181 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6241 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6301 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6361 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6421 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6481 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6541 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6601 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6661 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6721 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     6781 ccgtattacc gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata
     6841 gcccatatat ggagttccg
//
