LOCUS       sequence-356610-        5619 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human GFAP fused to 3xFLAG tag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5619)
  AUTHORS   Lee SH, Nam TS, Kim KH, Kim JH, Yoon W, Heo SH, Kim MJ, Shin BA, 
            Perng MD, Choy HE, Jo J, Kim MK, Choi SY
  TITLE     Aggregation-prone GFAP mutation in Alexander disease validated using
            a zebrafish model.
  JOURNAL   BMC Neurol. 2017 Sep 7;17(1):175. doi: 10.1186/s12883-017-0938-7.
  PUBMED    28882119
REFERENCE   2  (bases 1 to 5619)
  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: "BMC 
            Neurol."; date: "2017-09-7"; volume: "17(1):175. doi"; pages: " 
            10.1186/s12883-017-0938-7"
COMMENT     Sequence Label: pSYC-176
FEATURES             Location/Qualifiers
     source          1..5619
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        67..1051
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1086..1104
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1086..1103
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             2465..2488
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2492..2515
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2519..2542
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2597..2653
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(2698..2717)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2699..2717)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2722..2856
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2746..2765)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2800..2819
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2969..2989)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2969..2987)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3008..3024)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3008..3024)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3021..3043)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3032..3048
                     /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(3056..3062,3063..3080,3081..3086))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3101..3122
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3239..3256)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3410..3998)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3490..3509)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4169..4960,4961..5029))
                     /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     4792..4811
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5030..5134)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5160..5615)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5297..5318)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5509..5528
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
       61 acgccagtcg accgccaatt caatatggcg tatatggact catgccaatt caatatggtg
      121 gatctggacc tgtgccaatt caatatggcg tatatggact cgtgccaatt caatatggtg
      181 gatctggacc ccagccaatt caatatggcg gacttggcac catgccaatt caatatggcg
      241 gacctggcac tgtgccaact ggggaggggt ctacttggca cggtgccaag tttgaggagg
      301 ggtcttggcc ctgtgccaag tccgccatat tgaattggca tggtgccaat aatggcggcc
      361 atattggcta tatgccagga tcaatatata ggcaatatcc aatatggccc tatgccaata
      421 tggctattgg ccaggttcaa tactatgtat tggccctatg ccatatagta ttccatatat
      481 gggttttcct attgacgtag atagcccctc ccaatgggcg gtcccatata ccatatatgg
      541 ggcttcctaa taccgcccat agccactccc ccattgacgt caatggtctc tatatatggt
      601 ctttcctatt gacgtcatat gggcggtcct attgacgtat atggcgcctc ccccattgac
      661 gtcaattacg gtaaatggcc cgcctggctc aatgcccatt gacgtcaata ggaccaccca
      721 ccattgacgt caatgggatg gctcattgcc cattcatatc cgttctcacg ccccctattg
      781 acgtcaatga cggtaaatgg cccacttggc agtacatcaa tatctattaa tagtaacttg
      841 gcaagtacat tactattgga agtacgccag ggtacattgg cagtactccc attgacgtca
      901 atggcggtaa atggcccgcg atggctgcca agtacatccc cattgacgtc aatggggagg
      961 ggcaatgacg caaatgggcg ttccattgac gtaaatgggc ggtaggcgtg cctaatggga
     1021 ggtctatata agcaatgctc gtttagggaa ccgccattct gcctggggac gtcggagcaa
     1081 gcttgattta ggtgacacta tagaatacaa gctacttgtt ctttttgcag gatccatgga
     1141 gaggagacgc atcacctccg ctgctcgccg ctcctacgtc tcctcagggg agatgatggt
     1201 ggggggcctg gctcctggcc gccgtctggg tcctggcacc cgcctctccc tggctcgaat
     1261 gccccctcca ctcccgaccc gggtggattt ctccctggct ggggcactca atgctggctt
     1321 caaggagacc cgggccagtg agcgggcaga gatgatggag ctcaatgacc gctttgccag
     1381 ctacatcgag aaggttcgct tcctggaaca gcaaaacaag gcgctggctg ctgagctgaa
     1441 ccagctgcgg gccaaggagc ccaccaagct ggcagacgtc taccaggctg agctgcgaga
     1501 gctgcggctg cggctcgatc aactcaccgc caacagcgcc cggctggagg ttgagaggga
     1561 caatctggca caggacctgg ccactgtgag gcagaagctc caggatgaaa ccaacctgag
     1621 gctggaagcc gagaacaacc tggctgccta tagacaggaa gcagatgaag ccaccctggc
     1681 ccgtctggat ctggagagga agattgagtc gctggaggag gagatccggt tcttgaggaa
     1741 gatccacgag gaggaggttc gggaactcca ggagcagctg gcccgacagc aggtccatgt
     1801 ggagcttgac gtggccaagc cagacctcac cgcagccctg aaagagatcc gcacgcagta
     1861 tgaggcaatg gcgtccagca acatgcatga agccgaagag tggtaccgct ccaagtttgc
     1921 agacctgaca gacgctgctg cccgcaacgc ggagctgctc cgccaggcca agcacgaagc
     1981 caacgactac cggcgccagt tgcagtcctt gacctgcgac ctggagtctc tgcgcggcac
     2041 gaacgagtcc ctggagaggc agatgcgcga gcaggaggag cggcacgtgc gggaggcggc
     2101 cagttatcag gaggcgctgg cgcggctgga ggaagagggg cagagcctca aggacgagat
     2161 ggcccgccac ttgcaggagt accaggacct gctcaatgtc aagctggccc tggacatcga
     2221 gatcgccacc tacaggaagc tgctagaggg cgaggagaac cggatcacca ttcccgtgca
     2281 gaccttctcc aacctgcaga ttcgagaaac cagcctggac accaagtctg tgtcagaagg
     2341 ccacctcaag aggaacatcg tggtgaagac cgtggagatg cgggatggag aggtcattaa
     2401 ggagtccaag caggagcaca aggatgtgat gatgatatct ggcgcgccta ccatggggcc
     2461 cggcgactac aaggatgacg atgacaaggg cgactacaag gatgacgatg acaagggcga
     2521 ctacaaggat gacgatgaca agggccggcc taagatcacc ggtcccgggc ttaaggagct
     2581 cgcatgcgga agcggagcta ctaacttcag cctgctgaag caggctggag acgtggagga
     2641 gaaccctgga cctagatctg gaattcaagg cctctcgagc ctctagatag ctagcctccc
     2701 tatagtgagt cgtattacgt agatccagac atgataagat acattgatga gtttggacaa
     2761 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     2821 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     2881 atgtttcagg ttcaggggga ggtgtgggag gttttttaat tcgcggccgc ggcgccaatg
     2941 cattgggccc ggtacccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     3001 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     3061 tacgagccgg gagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     3121 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3181 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3241 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3301 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3361 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3421 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3481 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3541 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3601 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3661 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3721 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3781 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3841 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3901 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3961 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4021 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4081 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4141 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4201 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4261 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4321 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4381 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4441 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4501 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4561 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4621 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4681 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4741 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4801 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4861 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4921 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4981 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5041 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5101 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     5161 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     5221 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     5281 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     5341 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     5401 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     5461 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     5521 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     5581 acccgccgcg cttaatgcgc cgctacaggg cgcgtccca
//
