LOCUS       sequence-362540-        5720 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Granulin 2 without linker 3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5720)
  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 5720)
  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 2-no linker
FEATURES             Location/Qualifiers
     source          1..5720
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(166..185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        357..736
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        737..940
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     891..911
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     985..1004
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        985..1003
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1096..1101,1102..1125,1126..1161,1162..1185)
                     /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             1195..1218
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1433..1450)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1439..1663
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1709..2137
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1796..1815)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2006..2027
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2146..2166)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2151..2480
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2331..2466
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2393..2412
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2547..3341
                     /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(2601..2620)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3211..3230
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3515..3636
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3552..3571)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3606..3625
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3685..3701)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3685..3701)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3698..3720)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3709..3725
                     /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(3733..3739,3740..3757,3758..3763))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3778..3799
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3916..3933)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4087..4675)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4167..4186)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4846..5637,5638..5706))
                     /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     5469..5488
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5707..5720,1..91))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
       61 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
      121 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
      181 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      241 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      301 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      361 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      421 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      481 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      541 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      601 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      661 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      721 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      781 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      841 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      901 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      961 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta
     1021 gcgtttaaac ttaagcttgc caccatgtgg accctggtgt cctgggtcgc actgacagca
     1081 ggactggtgg ctggatctgc atggagtcac ccccagttcg agaagggagg aggatccgga
     1141 ggaggatcag gagggagcgc atggtcccat cctcagtttg aaaaaggcgc ctctgactac
     1201 aaggacgatg acgataaagc tatccagtgc cctgactctc agttcgagtg tccagacttc
     1261 agcacatgct gcgtgatggt cgatggatca tggggctgct gtcccatgcc tcaggccagc
     1321 tgctgtgaag acagggtgca ctgctgtcca catggcgcct tctgcgatct ggtccacact
     1381 cggtgtatta cctgactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact
     1441 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     1501 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     1561 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     1621 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     1681 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     1741 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1801 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1861 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1921 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1981 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2041 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2101 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     2161 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     2221 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2281 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     2341 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     2401 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     2461 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     2521 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     2581 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     2641 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     2701 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     2761 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     2821 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     2881 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     2941 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     3001 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     3061 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     3121 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     3181 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     3241 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     3301 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     3361 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     3421 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3481 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     3541 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3601 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     3661 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     3721 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     3781 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3841 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3901 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3961 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4021 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4081 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4141 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4201 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4261 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4321 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4381 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4441 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4501 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4561 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4621 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4681 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4741 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4801 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4861 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4921 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4981 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5041 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5101 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5161 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5221 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5281 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5341 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5401 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5461 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5521 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5581 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5641 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5701 actcatactc ttcctttttc
//
