LOCUS       sequence-345367-        7410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses WIPI2B (R108E) fused to Halo in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7410)
  AUTHORS   Stavoe AK, Gopal PP, Gubas A, Tooze SA, Holzbaur EL
  TITLE     Expression of WIPI2B counteracts age-related decline in 
            autophagosome biogenesis in neurons
  JOURNAL   Elife. 2019 Jul 16;8. pii: 44219. doi: 10.7554/eLife.44219.
  PUBMED    31309927
REFERENCE   2  (bases 1 to 7410)
  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: "Elife."; 
            date: "2019-07-16"; volume: "8. pii: 44219. doi"; pages: " 
            10.7554/eLife.44219"
COMMENT     Sequence Label: Halo-WIPI2B R108E
FEATURES             Location/Qualifiers
     source          1..7410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        150..529
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        530..733
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     684..704
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          869..1001
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     984..1003
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1045..1064
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1045..1063
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1073..1082
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1079..1969
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1982..2002
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(3373..3391)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3423..3544
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3434..3453)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3488..3507
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3789..4146
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3789..3980
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3812..3832)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3997..4132
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4197..4991
                     /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(4251..4270)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4861..4880
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5055..5103
                     /note="synthetic polyadenylation signal"
     promoter        5259..5363
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5364..5432,5433..6224)
                     /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     complement(5582..5601)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6382..6970)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6462..6481)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    7086..7369
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 ggatcgagat cttcaatatt ggccattagc catattattc attggttata tagcataaat
       61 caatattggc tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat
      121 tggctcatgt ccaatatgac cgccatgttg gcattgatta ttgactagtt attaatagta
      181 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      241 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      301 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      361 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat
      421 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga
      481 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      541 ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      601 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      661 tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      721 tataagcaga gctggtttag tgaaccgtca gatcactaga agctttattg cggtagttta
      781 tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga acttaagctg
      841 cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac aggtttaagg
      901 agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct gataggcacc
      961 tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact cccagttcaa
     1021 ttacagctct taaggctaga gtattaatac gactcactat agggctagca aagccaccat
     1081 ggcagaaatc ggtactggct ttccattcga cccccattat gtggaagtcc tgggcgagcg
     1141 catgcactac gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa
     1201 cccgacctcc tcctacgtgt ggcgcaacat catcccgcat gttgcaccga cccatcgctg
     1261 cattgctcca gacctgatcg gtatgggcaa atccgacaaa ccagacctgg gttatttctt
     1321 cgacgaccac gtccgcttca tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt
     1381 cctggtcatt cacgactggg gctccgctct gggtttccac tgggccaagc gcaatccaga
     1441 gcgcgtcaaa ggtattgcat ttatggagtt catccgccct atcccgacct gggacgaatg
     1501 gccagaattt gcccgcgaga ccttccaggc cttccgcacc accgacgtcg gccgcaagct
     1561 gatcatcgat cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct
     1621 gactgaagtc gagatggacc attaccgcga gccgttcctg aatcctgttg accgcgagcc
     1681 actgtggcgc ttcccaaacg agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct
     1741 ggtcgaagaa tacatggact ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg
     1801 caccccaggc gttctgatcc caccggccga agccgctcgc ctggccaaaa gcctgcctaa
     1861 ctgcaaggct gtggacatcg gcccgggtct gaatctgctg caagaagaca acccggacct
     1921 gatcggcagc gagatcgcgc gctggctgtc gacgctcgag atttccggcg agccaaccac
     1981 tgaggatctg tactttcaga gcgataacgc gatcgcttcc gaattcggat ccatgaacct
     2041 ggcgagccag agcggggagg ccggcgccgg ccagctgctc ttcgccaact tcaaccagga
     2101 caacacgtcc ctagctgttg gtagtaagtc cggttataaa tttttctccc tttcttctgt
     2161 ggataagctg gaacagatct atgaatgcac cgatacggaa gatgtgtgca ttgtagagag
     2221 attgttctcc agcagcctag tggccatcgt gagccttaaa gcaccaagga agctaaaggt
     2281 ttgccacttt aagaagggaa ctgagatctg caactacagc tactccaaca cgattctggc
     2341 tgtgaagctc aacgagcaga ggctgatagt atgcctggag gagtccctgt acatccacaa
     2401 cattcgggac atgaaggtgc tgcatacgat cagggagacg cctccaaacc ctgcaggcct
     2461 gtgtgcgctg tcaatcaaca acgacaactg ctacttggcg tacccaggga gcgcgaccat
     2521 cggagaggtg caggtcttcg ataccattaa tttgagagct gcaaacatga ttccggctca
     2581 cgacagtcct ttagcggcac tggcctttga cgcaagtgga actaaacttg ccacggcttc
     2641 ggagaagggg accgtgatta gggtattttc cattccagaa ggacaaaaac tctttgagtt
     2701 tcggagagga gtaaagaggt gcgtgagcat ctgctccctg gccttcagca tggacggcat
     2761 gttcctctcc gcctccagca acactgagac cgtgcacatc ttcaaactcg agactgtgaa
     2821 agaaaaaccc ccagaggagc ccaccacctg gaccgggtac ttcgggaaag tgctcatggc
     2881 ctccaccagc tacctgcctt cccaagtgac agaaatgttc aaccagggca gagccttcgc
     2941 cacggtccgc ctgccattct gcggccacaa aaacatctgc tcgctagcca caattcagaa
     3001 gatcccgcgg ttgttggtgg gtgccgccga cgggtacctg tacatgtaca acctggaccc
     3061 ccaggagggc ggcgagtgtg ccctgatgaa gcagcaccgg ctggacggca gtctggaaac
     3121 gaccaatgag atcttggact ctgcctctca cgactgcccc ttagtcactc agacatacgg
     3181 cgcagctgca ggaaaaggta cttacgtgcc ttcatcccca acgagacttg cctacacaga
     3241 cgacctgggt gctgtgggtg gcgcctgcct ggaggacgag gccagcgccc tgcgcctgga
     3301 tgaggacagc gagcacccgc ccatgattct tcggactgac tgagcggccg cttgctgagt
     3361 tggctgctgc caccgctgag caataactag cataacccct tggccgcttc gagcagacat
     3421 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     3481 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     3541 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt
     3601 ttttttaagc aagtaaaacc tctacaaatg tggtaaaatc gaattttaac aaaatattaa
     3661 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3721 catacgcgga tctgcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta
     3781 ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     3841 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3901 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3961 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4021 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4081 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4141 tgcaaaaagc ttgattcttc tgacacaaca gtctcgaact taaggctaga gccaccatga
     4201 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     4261 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     4321 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     4381 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     4441 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     4501 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     4561 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     4621 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     4681 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     4741 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     4801 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     4861 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     4921 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4981 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     5041 atcacgatgg ccgcaataaa atatctttat tttcattaca tctgtgtgtt ggttttttgt
     5101 gtgaatcgat agcgataagg atcctctttg cgcttgcgtt ttcccttgtc cagatagccc
     5161 agtagctgac attcatccgg ggtcagcacc gtttctgcgg actggctttc tacgtaatgg
     5221 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5281 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5341 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5401 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5461 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5521 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact ttcaaagttc
     5581 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5641 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5701 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5761 ccaacttact tctgacaact atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5821 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     5881 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     5941 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6001 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6061 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6121 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6181 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaattcgaa atgaccgacc
     6241 aagcgacgcc caaccggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     6301 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     6361 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6421 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6481 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6541 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6601 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6661 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6721 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6781 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     6841 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6901 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6961 aggatttcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     7021 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     7081 atagtccgga aatacaggaa cgcacgctgg atggcccttc gctgggatgg tgaaaccatg
     7141 aaaaatggca gcttcagtgg attaagtggg ggtaatgtgg cctgtaccct ctggttgcat
     7201 aggtattcat acggttaaaa tttatcaggc gcgattgcgg cagtttttcg ggtggtttgt
     7261 tgccattttt acctgtctgc tgccgtgatc gcgctgaacg cgttttagcg gtgcgtacaa
     7321 ttaagggatt atggtaaatc cacttactgt ctgccctcgt agccatcgag ataaaccgca
     7381 gtactccggc cacgatgcgt ccggcgtaga
//
