LOCUS       sequence-333372-        7544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  We generated a human-GFAP-EGFP fusion construct by randomly 
            inserting the coding region of EFGP into the coding region of 
            hGFAP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7544)
  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 7544)
  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: human-GFAP-EGFP
FEATURES             Location/Qualifiers
     source          1..7544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             165..187
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(232..251)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(233..251)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(296..499)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(325..345)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        500..879
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1051..1070
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1145..1249
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1250..1318,1319..2110)
                     /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     complement(1468..1487)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2281..2869
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2770..2789
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3023..3040
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3157..3178
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3193..3198,3199..3216,3217..3223)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3231..3247
                     /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)."
     primer_bind     3236..3258
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3255..3271
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3255..3271
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3320..3441
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3331..3350)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3385..3404
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3615..4409)
                     /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(3726..3745)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4336..4355
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4476..4805)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4490..4625
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4544..4563)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     4790..4810
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(4819..5247)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4929..4950)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5141..5160
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5293..5517
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5506..5523
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     misc_feature    6770..6788
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     CDS             complement(6806..7516)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             complement(6806..7516)
                     /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(6848..6869)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     7197..7216
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7456..7477
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 gtataagaga cagggtcggg agtggagggg gcattcgagc cagggagagg cgggtgccag
       61 gacccagacg gcggccagga gccaggcccc ccaccatcat ctcccctgag gagacgtagg
      121 agcggcgagc agcggaggtg atgcgtctcc tctccatatg tatatctcct tcttaaagtt
      181 aaacaaaatt atttctagag ggcccgttta aacgctagcc agcttgggtc tccctatagt
      241 gagtcgtatt aatttcgata agccagtaag cagtgggttc tctagttagc cagagagctc
      301 tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt
      361 tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt
      421 caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt
      481 actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag
      541 taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat
      601 tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag
      661 tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg
      721 gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc
      781 catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg
      841 taattgatta ctattaataa ctagtcaata atcaatgtca acgcgtatat ctggcccgta
      901 catcgcgaag cagcgcaaaa cgcctaaccc taagcagatt cttcatgcaa ttgtcggtca
      961 agccttgcct tgttgtagct taaattttgc tcgcgcacta ctcagcgacc tccaacacac
     1021 aagcagggag cagatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     1081 accatagggg atcgggagat ctcccgatcc gtcgacgtca ggtggcactt ttcggggaaa
     1141 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1201 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1261 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     1321 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     1381 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     1441 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     1501 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     1561 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     1621 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     1681 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     1741 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     1801 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     1861 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     1921 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     1981 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2041 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2101 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     2161 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     2221 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2281 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2341 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2401 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     2461 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2521 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2581 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2641 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2701 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2761 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2821 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2881 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     2941 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3001 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     3061 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     3121 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
     3181 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     3241 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     3301 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     3361 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     3421 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     3481 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     3541 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa
     3601 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     3661 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     3721 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     3781 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     3841 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac
     3901 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     3961 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     4021 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     4081 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     4141 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     4201 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
     4261 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     4321 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     4381 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     4441 gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag gcctccaaaa
     4501 aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc ctctgcataa
     4561 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     4621 gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc
     4681 atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga
     4741 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga
     4801 cacacattcc acagaattaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4861 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4921 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4981 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     5041 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     5101 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     5161 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     5221 cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag ctggttcttt
     5281 ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg tcttcccaat
     5341 cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac
     5401 aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg
     5461 tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacagtcg
     5521 aggctgatca gcggtttaaa cttaagcttg gtaccgagct cggatccact agtccagtgt
     5581 ggtggaattc gatttgggtc ctgcctcaca tcacatcctt gtgctcctgc ttggactcct
     5641 taatgacctc tccatcccgc atctccacgg tcttcaccac gatgttcctc ttgaggtggc
     5701 cttctgacac agacttggtg tccaggctgg tttctcgaat ctgcaggttg gagaaggtct
     5761 gcacgggaat ggtgatccgg ttctcctcgc cctctagcag cttcctgtag gtggcgatct
     5821 cgatgtccag ggccagcttg acattgagca ggtcctggta ctcctgcaag tggcgggcca
     5881 tctcgtcctt gaggctctgc ccctcttcct ccagccgcgc cagcgcctcc tgataactgg
     5941 ccgcctcccg cacgtgccgc tcctcctgct cgcgcatctg cctctccagg gactcgttcg
     6001 tgccgcgcag agactccagg tcgcaggtca aggactgcaa ctggcgccgg tagtcgttgg
     6061 cttcgtgctt ggcctggcgg agcagctccg cgttgcgggc agcagcgtct gtcaggtctg
     6121 caaacttgga gcggtaccac tcttcggctt catgcatgtt gctggacgcc attgcctcat
     6181 actgcgtgcg gatctctttc agggctgcgg tgaggtctgg cttggccacg tcaagctcca
     6241 catggacctg ctgtcgggcc agctgctcct ggagttcccg aacctcctcc tcgtggatct
     6301 tcctcaagaa ccggatctcc tcctccagcg actcaatctt cctctccaga tccagacggg
     6361 ccagggtggc ttcatctgct tcctgtctat aggcagccag gttgttctcg gcttccagcc
     6421 tcaggttggt ttcatcctgg agcttctgcc tcacagtggc caggtcctgt gccagattgt
     6481 ccctctcaac ctccagccgg gcgctgttgg cggtgagttg atcgagccgc agccgcagct
     6541 ctcgcagctc agcctggtag acgtctgcca gcttggtggg ctccttggcc cgcagctggt
     6601 tcagctcagc agccagcgcc ttgttttgct gttccaggaa gcgaaccttc tcgatgtagc
     6661 tggcaaagcg gtcattgagc tccatcatct ctgcccgctc actggcccgg gtctccttga
     6721 agccagcatt gagtgcccca gccagggaga aatccacccg ggtcgggagc tgtctcttat
     6781 acacatctgc ccgggcgcgc ccacccttgt acagctcgtc catgccgaga gtgatcccgg
     6841 cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttgctca
     6901 gggcggactg ggtgctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg
     6961 gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga
     7021 tcttgaagtt caccttgatg ccgttcttct gcttgtcggc catgatatag acgttgtggc
     7081 tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc
     7141 ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct
     7201 tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg
     7261 cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc
     7321 cgtaggtcag ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga
     7381 tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga
     7441 acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca
     7501 gctcctcgcc cttgctggcg cgccagatgt gtataagcgg ggtc
//
