LOCUS       sequence-347172-        5714 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 5714)
  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 5714)
  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..5714
                     /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(1071..1088)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1077..1301
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1347..1775
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1434..1453)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1644..1665
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1784..1804)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1789..2118
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1969..2104
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2031..2050
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2185..2979
                     /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(2239..2258)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2849..2868
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3153..3274
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3190..3209)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3244..3263
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3323..3339)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3323..3339)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3336..3358)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3347..3363
                     /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(3371..3377,3378..3395,3396..3401))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3416..3437
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3554..3571)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3725..4313)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3805..3824)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4484..5275,5276..5344))
                     /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     5107..5126
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5345..5449)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5524..5543)
                     /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 gtgagctggg tcgccctgac
      721 cgctggactg gtggcaggat ccgcctggtc tcacccccag ttcgagaagg gaggaggaag
      781 tggcggcgga agtggaggat cagcctggag ccatcctcag tttgaaaaag gcgcttctga
      841 ctacaaggac gatgacgata aagacgtgcc atgcgataac gtcagctcct gtccctctag
      901 tgatacctgc tgtcagctga catccggaga gtggggctgc tgtcccatcc ctgaagccgt
      961 gtgctgttct gaccaccagc attgctgtcc ccagggatat acatgcgtgg cagagggcca
     1021 gtgtcagagg tgactcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     1081 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     1141 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     1201 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     1261 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     1321 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     1381 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     1441 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     1501 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     1561 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     1621 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     1681 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     1741 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     1801 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     1861 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     1921 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     1981 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     2041 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     2101 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     2161 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     2221 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     2281 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     2341 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     2401 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     2461 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     2521 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     2581 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     2641 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     2701 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     2761 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     2821 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     2881 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     2941 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     3001 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     3061 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     3121 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     3181 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     3241 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     3301 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     3361 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     3421 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     3481 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     3541 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3601 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3661 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3721 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3781 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3841 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3901 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3961 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4021 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4081 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4141 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     4201 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4261 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4321 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4381 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     4441 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     4501 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     4561 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     4621 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     4681 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4741 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4801 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4861 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4921 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4981 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5041 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5101 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5161 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5221 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5281 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     5341 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     5401 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     5461 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     5521 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     5581 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     5641 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     5701 cagatatacg cgtt
//
