LOCUS       sequence-333373-        7537 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single p.Arg239Cys(R239C) mutations was individually inserted by 
            site-directed mutagenesis into the wild-type hGFAP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7537)
  AUTHORS   Letra-Vilela R, Quiteres R, Murtinheira F, Crevenna A, Hensel Z, 
            Herrera F
  TITLE     New tools for the visualization of glial fibrillary acidic protein 
            in living cells
  JOURNAL   Experimental Results (2020), Volume 1, e4, 1–8
REFERENCE   2  (bases 1 to 7537)
  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: 
            "Experimental Results (2020), Volume 1, e4, 1–8"
COMMENT     Sequence Label: hGFAP R239C
FEATURES             Location/Qualifiers
     source          1..7537
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             21..731
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             21..731
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(60..81)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(321..340)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     668..689
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    749..767
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     primer_bind     complement(2014..2031)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2020..2244
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2290..2718
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2377..2396)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2587..2608
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2727..2747)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2732..3061
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2912..3047
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2974..2993
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3128..3922
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3182..3201)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3792..3811
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4096..4217
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4133..4152)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4187..4206
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4266..4282)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4266..4282)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4279..4301)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4290..4306
                     /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(4314..4320,4321..4338,4339..4344))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4359..4380
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4497..4514)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4668..5256)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4748..4767)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5427..6218,6219..6287))
                     /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     6050..6069
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6288..6392)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6467..6486)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6658..7037
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7038..7241
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7192..7212
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     7286..7305
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7286..7304
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             7350..7372
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ttatacacat ctggcgcgcc agcaagggcg aggagctgtt caccggggtg gtgcccatcc
       61 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      121 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      181 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      241 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      301 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
      361 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
      421 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
      481 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
      541 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
      601 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
      661 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
      721 agctgtacaa gggtgggcgc gcccgggcag atgtgtataa gagacagctc ccgacccggg
      781 tggatttctc cctggctggg gcactcaatg ctggcttcaa ggagacccgg gccagtgagc
      841 gggcagagat gatggagctc aatgaccgct ttgccagcta catcgagaag gttcgcttcc
      901 tggaacagca aaacaaggcg ctggctgctg agctgaacca gctgcgggcc aaggagccca
      961 ccaagctggc agacgtctac caggctgagc tgcgagagct gcggctgcgg ctcgatcaac
     1021 tcaccgccaa cagcgcccgg ctggaggttg agagggacaa tctggcacag gacctggcca
     1081 ctgtgaggca gaagctccag gatgaaacca acctgaggct ggaagccgag aacaacctgg
     1141 ctgcctatag acaggaagca gatgaagcca ccctggcccg tctggatctg gagaggaaga
     1201 ttgagtcgct ggaggaggag atccggttct tgaggaagat ccacgaggag gaggttcggg
     1261 aactccagga gcagctggcc cgacagcagg tccatgtgga gcttgacgtg gccaagccag
     1321 acctcaccgc agccctgaaa gagatctgca cgcagtatga ggcaatggcg tccagcaaca
     1381 tgcatgaagc cgaagagtgg taccgctcca agtttgcaga cctgacagac gctgctgccc
     1441 gcaacgcgga gctgctccgc caggccaagc acgaagccaa cgactaccgg cgccagttgc
     1501 agtccttgac ctgcgacctg gagtctctgc gcggcacgaa cgagtccctg gagaggcaga
     1561 tgcgcgagca ggaggagcgg cacgtgcggg aggcggccag ttatcaggag gcgctggcgc
     1621 ggctggagga agaggggcag agcctcaagg acgagatggc ccgccacttg caggagtacc
     1681 aggacctgct caatgtcaag ctggccctgg acatcgagat cgccacctac aggaagctgc
     1741 tagagggcga ggagaaccgg atcaccattc ccgtgcagac cttctccaac ctgcagattc
     1801 gagaaaccag cctggacacc aagtctgtgt cagaaggcca cctcaagagg aacatcgtgg
     1861 tgaagaccgt ggagatgcgg gatggagagg tcattaagga gtccaagcag gagcacaagg
     1921 atgtgatgtg aggcaggacc caaatcgaat tccaccacac tggactagtg gatccgagct
     1981 cggtaccaag cttaagttta aaccgctgat cagcctcgac tgtgccttct agttgccagc
     2041 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     2101 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     2161 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     2221 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     2281 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2341 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2401 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2461 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2521 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2581 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2641 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2701 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     2761 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     2821 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2881 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     2941 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3001 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3061 aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc
     3121 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     3181 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     3241 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     3301 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     3361 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     3421 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     3481 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     3541 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     3601 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     3661 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     3721 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     3781 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     3841 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     3901 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     3961 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     4021 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     4081 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4141 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4201 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     4261 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4321 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4381 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4441 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4501 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4561 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4621 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4681 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4741 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4801 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4861 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4921 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4981 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5041 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5101 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5161 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5221 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5281 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5341 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5401 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5461 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5521 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5581 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5641 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5701 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5761 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5821 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5881 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5941 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6001 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6061 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6121 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6181 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6241 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6301 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6361 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6421 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     6481 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     6541 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     6601 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
     6661 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     6721 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     6781 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     6841 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     6901 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     6961 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     7021 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     7081 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     7141 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     7201 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac
     7261 ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct
     7321 agcgtttaaa cgggccctct agaaataatt ttgtttaact ttaagaagga gatatacata
     7381 tggagaggag acgcatcacc tccgctgctc gccgctccta cgtctcctca ggggagatga
     7441 tggtgggggg cctggctcct ggccgccgtc tgggtcctgg cacccgcctc tccctggctc
     7501 gaatgccccc tccactcccg accctgtctc ttatacc
//
