LOCUS       sequence-345069-        7410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses WIPI2B (S395A) 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 S395A
FEATURES             Location/Qualifiers
     source          1..7410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        158..537
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        538..741
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     692..712
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          877..1009
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     992..1011
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1053..1072
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1053..1071
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1081..1090
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1087..1977
                     /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             1990..2010
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(3381..3399)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3431..3552
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3442..3461)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3496..3515
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3797..4154
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3797..3988
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3820..3840)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4005..4140
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4205..4999
                     /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(4259..4278)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4869..4888
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5063..5111
                     /note="synthetic polyadenylation signal"
     promoter        5267..5371
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5372..5440,5441..6232)
                     /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(5590..5609)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6390..6978)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6470..6489)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    7094..7377
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 ggcgtagagg atcgagatct tcaatattgg ccattagcca tattattcat tggttatata
       61 gcataaatca atattggcta ttggccattg catacgttgt atctatatca taatatgtac
      121 atttatattg gctcatgtcc aatatgaccg ccatgttggc attgattatt gactagttat
      181 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      241 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      301 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      361 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtccg
      421 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      481 ttacgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      541 atgcggtttt ggcagtacac caatgggcgt ggatagcggt ttgactcacg gggatttcca
      601 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      661 ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg
      721 gaggtctata taagcagagc tggtttagtg aaccgtcaga tcactagaag ctttattgcg
      781 gtagtttatc acagttaaat tgctaacgca gtcagtgctt ctgacacaac agtctcgaac
      841 ttaagctgca gaagttggtc gtgaggcact gggcaggtaa gtatcaaggt tacaagacag
      901 gtttaaggag accaatagaa actgggcttg tcgagacaga gaagactctt gcgtttctga
      961 taggcaccta ttggtcttac tgacatccac tttgcctttc tctccacagg tgtccactcc
     1021 cagttcaatt acagctctta aggctagagt attaatacga ctcactatag ggctagcaaa
     1081 gccaccatgg cagaaatcgg tactggcttt ccattcgacc cccattatgt ggaagtcctg
     1141 ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg
     1201 cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc
     1261 catcgctgca ttgctccaga cctgatcggt atgggcaaat ccgacaaacc agacctgggt
     1321 tatttcttcg acgaccacgt ccgcttcatg gatgccttca tcgaagccct gggtctggaa
     1381 gaggtcgtcc tggtcattca cgactggggc tccgctctgg gtttccactg ggccaagcgc
     1441 aatccagagc gcgtcaaagg tattgcattt atggagttca tccgccctat cccgacctgg
     1501 gacgaatggc cagaatttgc ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc
     1561 cgcaagctga tcatcgatca gaacgttttt atcgagggta cgctgccgat gggtgtcgtc
     1621 cgcccgctga ctgaagtcga gatggaccat taccgcgagc cgttcctgaa tcctgttgac
     1681 cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc
     1741 gtcgcgctgg tcgaagaata catggactgg ctgcaccagt cccctgtccc gaagctgctg
     1801 ttctggggca ccccaggcgt tctgatccca ccggccgaag ccgctcgcct ggccaaaagc
     1861 ctgcctaact gcaaggctgt ggacatcggc ccgggtctga atctgctgca agaagacaac
     1921 ccggacctga tcggcagcga gatcgcgcgc tggctgtcga cgctcgagat ttccggcgag
     1981 ccaaccactg aggatctgta ctttcagagc gataacgcga tcgcttccga attcggatcc
     2041 atgaacctgg cgagccagag cggggaggcc ggcgccggcc agctgctctt cgccaacttc
     2101 aaccaggaca acacgtccct agctgttggt agtaagtccg gttataaatt tttctccctt
     2161 tcttctgtgg ataagctgga acagatctat gaatgcaccg atacggaaga tgtgtgcatt
     2221 gtagagagat tgttctccag cagcctagtg gccatcgtga gccttaaagc accaaggaag
     2281 ctaaaggttt gccactttaa gaagggaact gagatctgca actacagcta ctccaacacg
     2341 attctggctg tgaagctcaa caggcagagg ctgatagtat gcctggagga gtccctgtac
     2401 atccacaaca ttcgggacat gaaggtgctg catacgatca gggagacgcc tccaaaccct
     2461 gcaggcctgt gtgcgctgtc aatcaacaac gacaactgct acttggcgta cccagggagc
     2521 gcgaccatcg gagaggtgca ggtcttcgat accattaatt tgagagctgc aaacatgatt
     2581 ccggctcacg acagtccttt agcggcactg gcctttgacg caagtggaac taaacttgcc
     2641 acggcttcgg agaaggggac cgtgattagg gtattttcca ttccagaagg acaaaaactc
     2701 tttgagtttc ggagaggagt aaagaggtgc gtgagcatct gctccctggc cttcagcatg
     2761 gacggcatgt tcctctccgc ctccagcaac actgagaccg tgcacatctt caaactcgag
     2821 actgtgaaag aaaaaccccc agaggagccc accacctgga ccgggtactt cgggaaagtg
     2881 ctcatggcct ccaccagcta cctgccttcc caagtgacag aaatgttcaa ccagggcaga
     2941 gccttcgcca cggtccgcct gccattctgc ggccacaaaa acatctgctc gctagccaca
     3001 attcagaaga tcccgcggtt gttggtgggt gccgccgacg ggtacctgta catgtacaac
     3061 ctggaccccc aggagggcgg cgagtgtgcc ctgatgaagc agcaccggct ggacggcagt
     3121 ctggaaacga ccaatgagat cttggactct gcctctcacg actgcccctt agtcactcag
     3181 acatacggcg cagctgcagg aaaaggtact tacgtgcctt cagccccaac gagacttgcc
     3241 tacacagacg acctgggtgc tgtgggtggc gcctgcctgg aggacgaggc cagcgccctg
     3301 cgcctggatg aggacagcga gcacccgccc atgattcttc ggactgactg agcggccgct
     3361 tgctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gccgcttcga
     3421 gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     3481 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     3541 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg
     3601 tgggaggttt ttttaagcaa gtaaaacctc tacaaatgtg gtaaaatcga attttaacaa
     3661 aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3721 tcacaccgca tacgcggatc tgcgcagcac catggcctga aataacctct gaaagaggaa
     3781 cttggttagg taccttctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
     3841 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3901 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3961 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     4021 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4081 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4141 taggcttttg caaaaagctt gattcttctg acacaacagt ctcgaactta aggctagagc
     4201 caccatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     4261 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     4321 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     4381 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4441 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4501 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4561 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4621 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4681 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     4741 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4801 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4861 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4921 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4981 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     5041 aacctgccat cacgatggcc gcaataaaat atctttattt tcattacatc tgtgtgttgg
     5101 ttttttgtgt gaatcgatag cgataaggat cctctttgcg cttgcgtttt cccttgtcca
     5161 gatagcccag tagctgacat tcatccgggg tcagcaccgt ttctgcggac tggctttcta
     5221 cgtaatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5281 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     5341 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     5401 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     5461 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     5521 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     5581 caaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     5641 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     5701 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5761 cactgcggcc aacttacttc tgacaactat cggaggaccg aaggagctaa ccgctttttt
     5821 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     5881 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     5941 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     6001 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     6061 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     6121 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     6181 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aattcgaaat
     6241 gaccgaccaa gcgacgccca accggtatca gctcactcaa aggcggtaat acggttatcc
     6301 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6361 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6421 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6481 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6541 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6601 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6661 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6721 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6781 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     6841 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6901 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6961 agaaaaaaag gatttcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     7021 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     7081 atccttttat agtccggaaa tacaggaacg cacgctggat ggcccttcgc tgggatggtg
     7141 aaaccatgaa aaatggcagc ttcagtggat taagtggggg taatgtggcc tgtaccctct
     7201 ggttgcatag gtattcatac ggttaaaatt tatcaggcgc gattgcggca gtttttcggg
     7261 tggtttgttg ccatttttac ctgtctgctg ccgtgatcgc gctgaacgcg ttttagcggt
     7321 gcgtacaatt aagggattat ggtaaatcca cttactgtct gccctcgtag ccatcgagat
     7381 aaaccgcagt actccggcca cgatgcgtcc
//
