LOCUS       sequence-343439-        5711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Granulin 5 without linker 6 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5711)
  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 5711)
  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 5-no linker
FEATURES             Location/Qualifiers
     source          1..5711
                     /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(1305..1322)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1311..1535
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1581..2009
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1668..1687)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1878..1899
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2018..2038)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2023..2352
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2203..2338
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2265..2284
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2419..3213
                     /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(2473..2492)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3083..3102
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3387..3508
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3424..3443)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3478..3497
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3557..3573)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3557..3573)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3570..3592)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3581..3597
                     /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(3605..3611,3612..3629,3630..3635))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3650..3671
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3788..3805)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3959..4544)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4039..4058)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4715..5506,5507..5575))
                     /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     5338..5357
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5576..5680)
                     /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 tctggtgagc tgggtcgccc tgaccgctgg
      961 actggtggca ggatccgcct ggtctcaccc ccagttcgag aagggaggag gaagtggcgg
     1021 cggaagtgga ggatcagcct ggagccatcc tcagtttgaa aaaggcgctt ctgactacaa
     1081 ggacgatgac gataaagacg tgccatgcga taacgtcagc tcctgtccct ctagtgatac
     1141 ctgctgtcag ctgacatccg gagagtgggg ctgctgtccc atccctgaag ccgtgtgctg
     1201 ttctgaccac cagcattgct gtccccaggg atatacatgc gtggcagagg gccagtgtca
     1261 gaggtgactc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     1321 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     1381 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     1441 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     1501 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     1561 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1621 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1681 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     1741 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1801 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1861 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1921 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1981 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     2041 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2101 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2161 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     2221 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     2281 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     2341 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     2401 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     2461 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     2521 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     2581 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     2641 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     2701 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     2761 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     2821 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     2881 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     2941 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     3001 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     3061 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     3121 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     3181 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     3241 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     3301 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     3361 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     3421 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3481 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     3541 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3601 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3661 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3721 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3781 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3841 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3901 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3961 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4021 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4081 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4141 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4201 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4261 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4321 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4381 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4441 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggttttt
     4501 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4561 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4621 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4681 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4741 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4801 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4861 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4921 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4981 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5041 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5101 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5161 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5221 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5281 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5341 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5401 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5461 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5521 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5581 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5641 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5701 cacctgacgt c
//
