LOCUS       sequence-362537-        5792 bp    DNA     circular SYN 14-APR-2025
DEFINITION  express Granulin 2 with linker 3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5792)
  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 5792)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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+linker3
FEATURES             Location/Qualifiers
     source          1..5792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(87..106)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        278..657
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        658..861
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     812..832
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     906..925
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        906..924
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1017..1022,1023..1046,1047..1082,1083..1106)
                     /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             1116..1139
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1426..1443)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1432..1656
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1702..2130
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     1999..2020
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2139..2159)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2144..2473
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2324..2459
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2386..2405
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2540..3334
                     /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(2594..2613)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3204..3223
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3508..3629
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3545..3564)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3599..3618
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3678..3694)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3678..3694)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3691..3713)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3702..3718
                     /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(3726..3732,3733..3750,3751..3756))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3771..3792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3909..3926)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4080..4668)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4160..4179)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4839..5630,5631..5699))
                     /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     5462..5481
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5700..5792,1..12))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
       61 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
      121 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
      181 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
      241 gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat
      301 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      361 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      421 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      481 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      541 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      601 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      661 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      721 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      781 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
      841 gaggtctata taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc
      901 gaaattaata cgactcacta tagggagacc caagctggct agcgtttaaa cttaagcttg
      961 ccaccatgtg gaccctggtg tcctgggtcg cactgacagc aggactggtg gctggatctg
     1021 catggagtca cccccagttc gagaagggag gaggatccgg aggaggatca ggagggagcg
     1081 catggtccca tcctcagttt gaaaaaggcg cctctgacta caaggacgat gacgataaag
     1141 ctatccagtg ccctgactct cagttcgagt gtccagactt cagcacatgc tgcgtgatgg
     1201 tcgatggatc atggggctgc tgtcccatgc ctcaggccag ctgctgtgaa gacagggtgc
     1261 actgctgtcc acatggcgcc ttctgcgatc tggtccacac tcggtgtatt accccaacag
     1321 ggactcatcc cctggccaag aaactgccag ctcagcgaac caacagagca gtggctctga
     1381 gctcctgact cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     1441 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     1501 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     1561 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     1621 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     1681 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1741 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1801 tcttcacttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1861 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1921 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1981 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2041 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2101 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     2161 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2221 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2281 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     2341 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     2401 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     2461 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     2521 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     2581 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     2641 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     2701 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     2761 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     2821 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     2881 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     2941 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     3001 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     3061 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     3121 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     3181 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     3241 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     3301 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     3361 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     3421 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     3481 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     3541 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     3601 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     3661 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3721 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3781 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3841 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     3901 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3961 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4021 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4081 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4141 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4201 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4261 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4321 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4381 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4441 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4501 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4561 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4621 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4681 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4741 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4801 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4861 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4921 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4981 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5041 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5101 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5161 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5221 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5281 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5341 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5401 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5461 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5521 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5581 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5641 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5701 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5761 atatttgaat gtatttagaa aaataaacaa at
//
