LOCUS       sequence-339418-        7410 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses WIPI2B 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 Apr 14, 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 WT
FEATURES             Location/Qualifiers
     source          1..7410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1033..1052
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1033..1051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1061..1070
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1067..1957
                     /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             1970..1990
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(3361..3379)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3411..3532
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3422..3441)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3476..3495
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3777..4134
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3777..3968
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3800..3820)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3985..4120
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4185..4979
                     /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(4239..4258)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4849..4868
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5043..5091
                     /note="synthetic polyadenylation signal"
     promoter        5247..5351
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5352..5420,5421..6212)
                     /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(5570..5589)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6370..6958)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6450..6469)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    7074..7357
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcaaa gccaccatgg cagaaatcgg
     1081 tactggcttt ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt
     1141 cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc
     1201 ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga
     1261 cctgatcggt atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt
     1321 ccgcttcatg gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca
     1381 cgactggggc tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg
     1441 tattgcattt atggagttca tccgccctat cccgacctgg gacgaatggc cagaatttgc
     1501 ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca
     1561 gaacgttttt atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga
     1621 gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt
     1681 cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata
     1741 catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt
     1801 tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt
     1861 ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga
     1921 gatcgcgcgc tggctgtcga cgctcgagat ttccggcgag ccaaccactg aggatctgta
     1981 ctttcagagc gataacgcga tcgcttccga attcggatcc atgaacctgg cgagccagag
     2041 cggggaggcc ggcgccggcc agctgctctt cgccaacttc aaccaggaca acacgtccct
     2101 agctgttggt agtaagtccg gttataaatt tttctccctt tcttctgtgg ataagctgga
     2161 acagatctat gaatgcaccg atacggaaga tgtgtgcatt gtagagagat tgttctccag
     2221 cagcctagtg gccatcgtga gccttaaagc accaaggaag ctaaaggttt gccactttaa
     2281 gaagggaact gagatctgca actacagcta ctccaacacg attctggctg tgaagctcaa
     2341 caggcagagg ctgatagtat gcctggagga gtccctgtac atccacaaca ttcgggacat
     2401 gaaggtgctg catacgatca gggagacgcc tccaaaccct gcaggcctgt gtgcgctgtc
     2461 aatcaacaac gacaactgct acttggcgta cccagggagc gcgaccatcg gagaggtgca
     2521 ggtcttcgat accattaatt tgagagctgc aaacatgatt ccggctcacg acagtccttt
     2581 agcggcactg gcctttgacg caagtggaac taaacttgcc acggcttcgg agaaggggac
     2641 cgtgattagg gtattttcca ttccagaagg acaaaaactc tttgagtttc ggagaggagt
     2701 aaagaggtgc gtgagcatct gctccctggc cttcagcatg gacggcatgt tcctctccgc
     2761 ctccagcaac actgagaccg tgcacatctt caaactcgag actgtgaaag aaaaaccccc
     2821 agaggagccc accacctgga ccgggtactt cgggaaagtg ctcatggcct ccaccagcta
     2881 cctgccttcc caagtgacag aaatgttcaa ccagggcaga gccttcgcca cggtccgcct
     2941 gccattctgc ggccacaaaa acatctgctc gctagccaca attcagaaga tcccgcggtt
     3001 gttggtgggt gccgccgacg ggtacctgta catgtacaac ctggaccccc aggagggcgg
     3061 cgagtgtgcc ctgatgaagc agcaccggct ggacggcagt ctggaaacga ccaatgagat
     3121 cttggactct gcctctcacg actgcccctt agtcactcag acatacggcg cagctgcagg
     3181 aaaaggtact tacgtgcctt catccccaac gagacttgcc tacacagacg acctgggtgc
     3241 tgtgggtggc gcctgcctgg aggacgaggc cagcgccctg cgcctggatg aggacagcga
     3301 gcacccgccc atgattcttc ggactgactg agcggccgct tgctgagttg gctgctgcca
     3361 ccgctgagca ataactagca taaccccttg gccgcttcga gcagacatga taagatacat
     3421 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     3481 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     3541 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt ttttaagcaa
     3601 gtaaaacctc tacaaatgtg gtaaaatcga attttaacaa aatattaacg cttacaattt
     3661 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgcggatc
     3721 tgcgcagcac catggcctga aataacctct gaaagaggaa cttggttagg taccttctga
     3781 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3841 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3901 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3961 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4021 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     4081 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt
     4141 gattcttctg acacaacagt ctcgaactta aggctagagc caccatgatt gaacaagatg
     4201 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4261 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4321 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     4381 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4441 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4501 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4561 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4621 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4681 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     4741 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4801 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4861 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4921 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4981 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgatggcc
     5041 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag
     5101 cgataaggat cctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat
     5161 tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtaatggtt tcttagacgt
     5221 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5281 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5341 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5401 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5461 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5521 gttttcgccc cgaagaacgt tttccaatga tgagcacttt caaagttctg ctatgtggcg
     5581 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5641 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5701 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5761 tgacaactat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5821 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     5881 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     5941 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6001 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6061 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6121 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6181 agataggtgc ctcactgatt aagcattggt aattcgaaat gaccgaccaa gcgacgccca
     6241 accggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     6301 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     6361 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     6421 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6481 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     6541 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6601 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6661 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6721 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6781 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     6841 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6901 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatttcaaga
     6961 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     7021 gattttggtc atgagattat caaaaaggat cttcacctag atccttttat agtccggaaa
     7081 tacaggaacg cacgctggat ggcccttcgc tgggatggtg aaaccatgaa aaatggcagc
     7141 ttcagtggat taagtggggg taatgtggcc tgtaccctct ggttgcatag gtattcatac
     7201 ggttaaaatt tatcaggcgc gattgcggca gtttttcggg tggtttgttg ccatttttac
     7261 ctgtctgctg ccgtgatcgc gctgaacgcg ttttagcggt gcgtacaatt aagggattat
     7321 ggtaaatcca cttactgtct gccctcgtag ccatcgagat aaaccgcagt actccggcca
     7381 cgatgcgtcc ggcgtagagg atcgagatct
//
