LOCUS       sequence-347173-        5777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Granulin 6 with linker 7 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5777)
  AUTHORS   Holler CJ, Taylor G, Deng Q, Kukar T
  TITLE     Intracellular Proteolysis of Progranulin Generates Stable, Lysosomal
            Granulins that Are Haploinsufficient in Patients with Frontotemporal
            Dementia Caused by GRN Mutations.
  JOURNAL   eNeuro. 2017 Aug 18;4(4). pii: eN-NWR-0100-17. doi: 
            10.1523/ENEURO.0100-17.2017. eCollection 2017 Jul-Aug.
  PUBMED    28828399
REFERENCE   2  (bases 1 to 5777)
  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: "eNeuro."; 
            date: "2017-08-18"; volume: "4(4). pii: eN-NWR-0100-17. doi"; pages:
            " 10.1523/ENEURO.0100-17.2017. eCollection 2017 Jul-Aug"
COMMENT     Sequence Label: pcDNA3.1(+)_Granulin 6+linker7
FEATURES             Location/Qualifiers
     source          1..5777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(740..745,746..769,770..805,806..829)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             839..862
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1134..1151)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1140..1364
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1410..1838
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1497..1516)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1707..1728
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1847..1867)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1852..2181
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2032..2167
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2094..2113
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2248..3042
                     /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(2302..2321)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2912..2931
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3216..3337
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3253..3272)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3307..3326
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3386..3402)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3386..3402)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3399..3421)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3410..3426
                     /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(3434..3440,3441..3458,3459..3464))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3479..3500
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3617..3634)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3788..4376)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3868..3887)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4547..5338,5339..5407))
                     /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     5170..5189
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5408..5512)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5587..5606)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacttaagc ttgccaccat gtggactctg gtgtcctggg tcgccctgac
      721 cgctggactg gtggcaggaa gcgcctggtc ccacccacag ttcgagaagg gaggaggatc
      781 tggaggagga tctggaggga gtgcttggtc acatcctcag tttgaaaaag gcgcctcaga
      841 ctacaaggac gatgacgata aagacatcgg ctgcgatcag cacaccagct gtcccgtggg
      901 gcagacatgc tgtcctagcc tgggaggatc ctgggcatgc tgtcagctgc ctcacgccgt
      961 gtgctgtgag gatcggcagc attgctgtcc agcaggatat acatgcaacg tcaaggccag
     1021 atcttgtgag aaagaagtgg tcagtgctca gccagcaacc ttcctggcaa ggtccccaca
     1081 tgtgggagtc aagtgactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     1141 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1201 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1261 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1321 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     1381 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     1441 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1501 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1561 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1621 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1681 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     1741 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     1801 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     1861 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     1921 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     1981 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2041 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     2101 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     2161 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     2221 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     2281 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     2341 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     2401 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     2461 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     2521 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     2581 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     2641 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     2701 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     2761 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     2821 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     2881 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     2941 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     3001 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     3061 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     3121 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     3181 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     3241 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3301 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     3361 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3421 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3481 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3541 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3601 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3661 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3721 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3781 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3841 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3901 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3961 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4021 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4081 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4141 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4201 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4261 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4321 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4381 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4441 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4501 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4561 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4621 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4681 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4741 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4801 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4861 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4921 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4981 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5041 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5101 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5161 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5221 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5281 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5341 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5401 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5461 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5521 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
     5581 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     5641 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     5701 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     5761 ggccagatat acgcgtt
//
