LOCUS       sequence-347167-        5774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Granulin 3 with linker 4 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5774)
  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 5774)
  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 3+linker4
FEATURES             Location/Qualifiers
     source          1..5774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(974..979,980..1003,1004..1039,1040..1063)
                     /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             1073..1096
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1365..1382)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1371..1595
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1641..2069
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1728..1747)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1938..1959
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2078..2098)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2083..2412
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2263..2398
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2325..2344
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2479..3273
                     /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(2533..2552)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3143..3162
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3447..3568
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3484..3503)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3538..3557
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3617..3633)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3617..3633)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3630..3652)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3641..3657
                     /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(3665..3671,3672..3689,3690..3695))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3710..3731
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3848..3865)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4019..4607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4099..4118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4778..5569,5570..5638))
                     /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     5401..5420
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5639..5743)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatgtggac actggtgtcc tgggtcgccc tgactgctgg
      961 actggtggca ggaagcgcct ggtcccaccc tcagttcgag aagggaggag gaagcggagg
     1021 aggatctgga ggaagtgctt ggtcacatcc acagtttgaa aaaggcgcat cagactacaa
     1081 ggacgatgac gataaaagcg tcatgtgccc agacgcccgg tctagatgtc ccgatgggag
     1141 tacctgctgt gagctgccct ccggaaagta tggctgctgt cccatgccta acgccacctg
     1201 ctgttctgac cacctgcatt gctgtcctca ggacacagtg tgcgatctga tccagagcaa
     1261 gtgtctgtcc aaagaaaatg ccaccacaga tctgctgact aaactgcccg ctcacaccgt
     1321 cggctgactc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     1381 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     1441 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     1501 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     1561 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     1621 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1681 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1741 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     1801 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1861 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1921 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1981 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     2041 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     2101 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2161 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2221 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     2281 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     2341 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     2401 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     2461 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     2521 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     2581 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     2641 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     2701 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     2761 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     2821 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     2881 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     2941 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     3001 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     3061 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     3121 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     3181 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     3241 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     3301 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     3361 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     3421 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     3481 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3541 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     3601 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3661 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3721 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3781 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3841 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3901 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3961 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4021 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4081 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4141 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4201 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4261 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4321 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4381 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4441 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4501 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4561 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4621 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4681 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4741 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4801 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4861 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4921 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4981 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5041 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5101 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5161 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5221 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5281 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5341 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5401 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5461 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5521 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5581 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5641 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5701 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5761 tgccacctga cgtc
//
