LOCUS       sequence-362539-        5741 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Granulin 1 with linker 2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5741)
  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 5741)
  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 1+linker2
FEATURES             Location/Qualifiers
     source          1..5741
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(75..94)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        266..645
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        646..849
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     800..820
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     894..913
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        894..912
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1005..1010,1011..1034,1035..1070,1071..1094)
                     /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             1104..1127
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1363..1380)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1369..1593
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1639..2067
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1726..1745)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1936..1957
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2076..2096)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2081..2410
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2261..2396
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2323..2342
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2477..3271
                     /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(2531..2550)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3141..3160
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3445..3566
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3482..3501)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3536..3555
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3615..3631)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3615..3631)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3628..3650)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3639..3655
                     /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(3663..3669,3670..3687,3688..3693))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3708..3729
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3846..3863)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4017..4605)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4097..4116)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4776..5567,5568..5636))
                     /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     5399..5418
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5637..5741)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
       61 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
      121 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
      181 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
      241 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
      301 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
      361 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      421 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      481 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
      541 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
      601 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
      661 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
      721 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      781 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      841 agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg
      901 actcactata gggagaccca agctggctag cgtttaaact taagcttgcc accatgtgga
      961 ctctggtgag ctgggtcgca ctgaccgcag gactggtggc tggatctgca tggagtcacc
     1021 ctcagttcga gaagggagga ggaagtggag gaggatcagg aggaagcgca tggtcccatc
     1081 cacagtttga aaaaggcgct tctgactaca aggacgatga cgataaagga ggcccctgcc
     1141 aggtcgatgc ccactgttct gctggccata gttgcatctt caccgtgtcc gggacaagct
     1201 cctgctgtcc ctttcctgag gccgtcgctt gtggggacgg acaccattgc tgtcccaggg
     1261 ggttccactg ttccgccgat ggacggtctt gctttcagag atcaggaaac aatagcgtgg
     1321 gctgactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     1381 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     1441 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     1501 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     1561 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     1621 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     1681 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     1741 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     1801 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     1861 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     1921 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     1981 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2041 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     2101 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2161 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2221 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     2281 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2341 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2401 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     2461 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     2521 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     2581 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     2641 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     2701 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     2761 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     2821 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     2881 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     2941 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     3001 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     3061 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     3121 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     3181 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     3241 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     3301 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3361 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3421 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     3481 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3541 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     3601 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3661 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3721 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3781 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3841 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3901 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3961 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4021 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4081 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4141 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4201 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4261 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4321 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4381 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4441 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4501 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4561 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4621 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4681 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4741 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4801 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4861 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4921 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4981 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5041 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5101 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5161 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5221 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5281 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5341 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5401 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5461 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5521 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5581 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5641 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5701 tttgaatgta tttagaaaaa taaacaaata ggggttccgc g
//
