LOCUS       sequence-345070-        7410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses WIPI2B (S395E) 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 S395E
FEATURES             Location/Qualifiers
     source          1..7410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        127..506
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        507..710
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     661..681
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          846..978
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     961..980
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1022..1041
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1022..1040
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1050..1059
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1056..1946
                     /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             1959..1979
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(3350..3368)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3400..3521
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3411..3430)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3465..3484
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3766..4123
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3766..3957
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3789..3809)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3974..4109
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4174..4968
                     /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(4228..4247)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4838..4857
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5032..5080
                     /note="synthetic polyadenylation signal"
     promoter        5236..5340
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5341..5409,5410..6201)
                     /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(5559..5578)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6359..6947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6439..6458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    7063..7346
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 cattagccat attattcatt ggttatatag cataaatcaa tattggctat tggccattgc
       61 atacgttgta tctatatcat aatatgtaca tttatattgg ctcatgtcca atatgaccgc
      121 catgttggca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      181 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      241 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      301 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      361 tacatcaagt gtatcatatg ccaagtccgc cccctattga cgtcaatgac ggtaaatggc
      421 ccgcctggca ttatgcccag tacatgacct tacgggactt tcctacttgg cagtacatct
      481 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacacc aatgggcgtg
      541 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      601 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taataacccc gccccgttga
      661 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga
      721 accgtcagat cactagaagc tttattgcgg tagtttatca cagttaaatt gctaacgcag
      781 tcagtgcttc tgacacaaca gtctcgaact taagctgcag aagttggtcg tgaggcactg
      841 ggcaggtaag tatcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcttgt
      901 cgagacagag aagactcttg cgtttctgat aggcacctat tggtcttact gacatccact
      961 ttgcctttct ctccacaggt gtccactccc agttcaatta cagctcttaa ggctagagta
     1021 ttaatacgac tcactatagg gctagcaaag ccaccatggc agaaatcggt actggctttc
     1081 cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat gcactacgtc gatgttggtc
     1141 cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc gacctcctcc tacgtgtggc
     1201 gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat tgctccagac ctgatcggta
     1261 tgggcaaatc cgacaaacca gacctgggtt atttcttcga cgaccacgtc cgcttcatgg
     1321 atgccttcat cgaagccctg ggtctggaag aggtcgtcct ggtcattcac gactggggct
     1381 ccgctctggg tttccactgg gccaagcgca atccagagcg cgtcaaaggt attgcattta
     1441 tggagttcat ccgccctatc ccgacctggg acgaatggcc agaatttgcc cgcgagacct
     1501 tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat catcgatcag aacgttttta
     1561 tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac tgaagtcgag atggaccatt
     1621 accgcgagcc gttcctgaat cctgttgacc gcgagccact gtggcgcttc ccaaacgagc
     1681 tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt cgaagaatac atggactggc
     1741 tgcaccagtc ccctgtcccg aagctgctgt tctggggcac cccaggcgtt ctgatcccac
     1801 cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg caaggctgtg gacatcggcc
     1861 cgggtctgaa tctgctgcaa gaagacaacc cggacctgat cggcagcgag atcgcgcgct
     1921 ggctgtcgac gctcgagatt tccggcgagc caaccactga ggatctgtac tttcagagcg
     1981 ataacgcgat cgcttccgaa ttcggatcca tgaacctggc gagccagagc ggggaggccg
     2041 gcgccggcca gctgctcttc gccaacttca accaggacaa cacgtcccta gctgttggta
     2101 gtaagtccgg ttataaattt ttctcccttt cttctgtgga taagctggaa cagatctatg
     2161 aatgcaccga tacggaagat gtgtgcattg tagagagatt gttctccagc agcctagtgg
     2221 ccatcgtgag ccttaaagca ccaaggaagc taaaggtttg ccactttaag aagggaactg
     2281 agatctgcaa ctacagctac tccaacacga ttctggctgt gaagctcaac aggcagaggc
     2341 tgatagtatg cctggaggag tccctgtaca tccacaacat tcgggacatg aaggtgctgc
     2401 atacgatcag ggagacgcct ccaaaccctg caggcctgtg tgcgctgtca atcaacaacg
     2461 acaactgcta cttggcgtac ccagggagcg cgaccatcgg agaggtgcag gtcttcgata
     2521 ccattaattt gagagctgca aacatgattc cggctcacga cagtccttta gcggcactgg
     2581 cctttgacgc aagtggaact aaacttgcca cggcttcgga gaaggggacc gtgattaggg
     2641 tattttccat tccagaagga caaaaactct ttgagtttcg gagaggagta aagaggtgcg
     2701 tgagcatctg ctccctggcc ttcagcatgg acggcatgtt cctctccgcc tccagcaaca
     2761 ctgagaccgt gcacatcttc aaactcgaga ctgtgaaaga aaaaccccca gaggagccca
     2821 ccacctggac cgggtacttc gggaaagtgc tcatggcctc caccagctac ctgccttccc
     2881 aagtgacaga aatgttcaac cagggcagag ccttcgccac ggtccgcctg ccattctgcg
     2941 gccacaaaaa catctgctcg ctagccacaa ttcagaagat cccgcggttg ttggtgggtg
     3001 ccgccgacgg gtacctgtac atgtacaacc tggaccccca ggagggcggc gagtgtgccc
     3061 tgatgaagca gcaccggctg gacggcagtc tggaaacgac caatgagatc ttggactctg
     3121 cctctcacga ctgcccctta gtcactcaga catacggcgc agctgcagga aaaggtactt
     3181 acgtgccttc agagccaacg agacttgcct acacagacga cctgggtgct gtgggtggcg
     3241 cctgcctgga ggacgaggcc agcgccctgc gcctggatga ggacagcgag cacccgccca
     3301 tgattcttcg gactgactga gcggccgctt gctgagttgg ctgctgccac cgctgagcaa
     3361 taactagcat aaccccttgg ccgcttcgag cagacatgat aagatacatt gatgagtttg
     3421 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     3481 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     3541 attttatgtt tcaggttcag ggggagatgt gggaggtttt tttaagcaag taaaacctct
     3601 acaaatgtgg taaaatcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt
     3661 attttctcct tacgcatctg tgcggtattt cacaccgcat acgcggatct gcgcagcacc
     3721 atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag gcggaaagaa
     3781 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3841 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     3901 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3961 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4021 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     4081 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagcttg attcttctga
     4141 cacaacagtc tcgaacttaa ggctagagcc accatgattg aacaagatgg attgcacgca
     4201 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     4261 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     4321 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     4381 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     4441 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     4501 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     4561 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     4621 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     4681 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     4741 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4801 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4861 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4921 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4981 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgatggccg caataaaata
     5041 tctttatttt cattacatct gtgtgttggt tttttgtgtg aatcgatagc gataaggatc
     5101 ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt
     5161 cagcaccgtt tctgcggact ggctttctac gtaatggttt cttagacgtc aggtggcact
     5221 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5281 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5341 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5401 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5461 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5521 gaagaacgtt ttccaatgat gagcactttc aaagttctgc tatgtggcgc ggtattatcc
     5581 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5641 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5701 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaactatc
     5761 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5821 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5881 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5941 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6001 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6061 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6121 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6181 tcactgatta agcattggta attcgaaatg accgaccaag cgacgcccaa ccggtatcag
     6241 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     6301 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     6361 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     6421 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     6481 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6541 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6601 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6661 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6721 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6781 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6841 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6901 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atttcaagaa gatcctttga
     6961 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     7021 tgagattatc aaaaaggatc ttcacctaga tccttttata gtccggaaat acaggaacgc
     7081 acgctggatg gcccttcgct gggatggtga aaccatgaaa aatggcagct tcagtggatt
     7141 aagtgggggt aatgtggcct gtaccctctg gttgcatagg tattcatacg gttaaaattt
     7201 atcaggcgcg attgcggcag tttttcgggt ggtttgttgc catttttacc tgtctgctgc
     7261 cgtgatcgcg ctgaacgcgt tttagcggtg cgtacaatta agggattatg gtaaatccac
     7321 ttactgtctg ccctcgtagc catcgagata aaccgcagta ctccggccac gatgcgtccg
     7381 gcgtagagga tcgagatctt caatattggc
//
