LOCUS       sequence-455753-        6300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Green Fluorescent Protein (GFP) with an N-terminal 
            twin-Strep tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6300)
  AUTHORS   Root J, Mendsaikhan A, Taylor G, Merino P, Nandy S, Wang M, Araujo 
            LT, Ryu D, Holler C, Thompson BM, Astarita G, Blain JF, Kukar T
  TITLE     Granulins rescue inflammation, lysosome dysfunction, lipofuscin, and
            neuropathology in a mouse model of progranulin deficiency.
  JOURNAL   Cell Rep. 2024 Nov 19;43(12):114985. doi: 
            10.1016/j.celrep.2024.114985.
  PUBMED    39565694
REFERENCE   2  (bases 1 to 6300)
  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: "Cell Rep.";
            date: "2024-11-19"; volume: "43(12):114985. doi"; pages: " 
            10.1016/j.celrep.2024.114985"
COMMENT     Sequence Label: pAAV GFP
FEATURES             Location/Qualifiers
     source          1..6300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   4..133
                     /label=AAV2 ITR
     promoter        439..716
                     /label=chicken beta-actin promoter
     primer_bind     995..1017
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1079..1098
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1189..1198
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1193..1202
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1210..1233
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1258..1281
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1297..1317
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1330..2043
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLCYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     FKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(1369..1390)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1977..1998
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2123..2711
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2176..2196)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2594..2605)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2738..2755)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2744..2968
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(3178..3507)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3192..3327
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3492..3512
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3753..4341)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3833..3852)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4512..5303,5304..5372))
                     /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     5135..5154
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5373..5477)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5545..5563
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5601..5623)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5723..5742
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5956..5975
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6265..6283
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6265..6282
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 tagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
       61 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
      121 actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
      181 tctaggtacc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      241 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      301 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      361 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      421 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
      481 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
      541 gggcgggggg gggggggggg cgcgcgccgg gggggggggg gggggggggg ggggggggsg
      601 gggsgrggcg gagaggkgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
      661 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
      721 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
      781 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
      841 gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc
      901 ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg
      961 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     1021 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     1081 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatca aagcttggta
     1141 ccgagctcgg atccactagt aacggccgcc agtgtgctgg aattcgccgc caccatggcg
     1201 gccgccggct ggagccaccc tcagttcgag aagggaggag gaggcggagg cggaggatgg
     1261 agccacccgc agttcgagaa aggagctagt ggcggagaga atctgtattt tcagggcgga
     1321 ggcggagcta gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1381 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1441 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1501 acactagtga ccaccctgtg ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     1561 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1621 ttcttcaagg acgacggcaa cttcaagacc cgcgccgagg tgaagttcga gggcgacacc
     1681 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1741 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     1801 aacggcatca aggttaactt caagacccgc cacaacatcg aggacggcag cgtgcagctc
     1861 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1921 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     1981 gtcctgctgg agttcgtgac cgccgccggc atcactcacg gcatggacga gctgtacaag
     2041 taaggatcca ctagtaacgg ccgccagtgt gctggaattc tgcagatatc gcggccgcac
     2101 tagtctcgag cagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac
     2161 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     2221 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     2281 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     2341 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     2401 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     2461 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     2521 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     2581 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     2641 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     2701 cgcctccccg catcgatacc gtcgactcgc tgatcagcct cgactgtgcc ttctagttgc
     2761 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2821 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     2881 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     2941 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg
     3001 actagagcat ggctacgtag ataagtagca tggcgggtta atcattaact acaaggaacc
     3061 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     3121 accaaaggtc gcccgacgcc cgggcggcct cagtgagcga gcgagcgcgc agagcttttt
     3181 gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga
     3241 ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc
     3301 ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
     3361 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     3421 cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg
     3481 ggactttcca caccctaact gacacacatt ccacagctgc attaatgaat cggccaacgc
     3541 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3601 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3661 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3721 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3781 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3841 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3901 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3961 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4021 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4081 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4141 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4201 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4261 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4321 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4381 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4441 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4501 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4561 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4621 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4681 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4741 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4801 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4861 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4921 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4981 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5041 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5101 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5161 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5221 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5281 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5341 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5401 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5461 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     5521 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt
     5581 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     5641 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     5701 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccattcg
     5761 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     5821 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     5881 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     5941 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     6001 ggtgatgccg gccacgatgc gtccggcgta gaggatctgg ctagcgatga ccctgctgat
     6061 tggttcgctg accatttccg ggtgcgggac ggcgttacca gaaactcaga aggttcgtcc
     6121 aaccaaaccg actctgacgg cagtttacga gagagatgat agggtctgct tcagtaagcc
     6181 agatgctaca caattaggct tgtacatatt gtcgttagaa cgcggctaca attaatacat
     6241 aaccttatgt atcatacaca tacgatttag gtgacactat agaatacacg gaattaattc
//
