LOCUS       sequence-350257-        7410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses WIPI2B (L69E) fused to Halo in mammalian cells to abrogate
            interaction with ATG16L1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7410)
  AUTHORS   Strong LM, Chang C, Riley JF, Boecker CA, Flower TG, Buffalo CZ, Ren
            X, Stavoe AK, Holzbaur EL, Hurley JH
  TITLE     Structural basis for membrane recruitment of ATG16L1 by WIPI2 in 
            autophagy.
  JOURNAL   Elife. 2021 Sep 10;10. pii: 70372. doi: 10.7554/eLife.70372.
  PUBMED    34505572
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: "2021-09-10"; volume: "10. pii: 70372. doi"; pages: " 
            10.7554/eLife.70372"
COMMENT     Sequence Label: Halo-WIPI2B L69E
FEATURES             Location/Qualifiers
     source          1..7410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..439
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        82..273
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(105..125)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      290..425
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             490..1284
                     /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(544..563)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1154..1173
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1348..1396
                     /note="synthetic polyadenylation signal"
     promoter        1552..1656
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1657..1725,1726..2517)
                     /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(1875..1894)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(2675..3263)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2755..2774)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    3379..3662
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     enhancer        3853..4232
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4233..4436
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4387..4407
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          4572..4704
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4687..4706
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     4748..4767
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4748..4766
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4776..4785
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4782..5672
                     /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             5685..5705
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(7076..7094)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    7126..7247
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7137..7156)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7191..7210
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 gtatttcaca ccgcatacgc ggatctgcgc agcaccatgg cctgaaataa cctctgaaag
       61 aggaacttgg ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
      121 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
      181 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
      241 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
      301 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
      361 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
      421 aggcctaggc ttttgcaaaa agcttgattc ttctgacaca acagtctcga acttaaggct
      481 agagccacca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
      541 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
      601 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
      661 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
      721 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
      781 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
      841 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
      901 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
      961 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     1021 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     1081 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     1141 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     1201 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     1261 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     1321 cgcccaacct gccatcacga tggccgcaat aaaatatctt tattttcatt acatctgtgt
     1381 gttggttttt tgtgtgaatc gatagcgata aggatcctct ttgcgcttgc gttttccctt
     1441 gtccagatag cccagtagct gacattcatc cggggtcagc accgtttctg cggactggct
     1501 ttctacgtaa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     1561 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     1621 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     1681 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     1741 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     1801 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     1861 actttcaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     1921 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     1981 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     2041 gataacactg cggccaactt acttctgaca actatcggag gaccgaagga gctaaccgct
     2101 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     2161 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     2221 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     2281 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     2341 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     2401 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     2461 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaattc
     2521 gaaatgaccg accaagcgac gcccaaccgg tatcagctca ctcaaaggcg gtaatacggt
     2581 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2641 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2701 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2761 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2821 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2881 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2941 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3001 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3061 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     3121 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3181 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3241 cgcgcagaaa aaaaggattt caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3301 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3361 cctagatcct tttatagtcc ggaaatacag gaacgcacgc tggatggccc ttcgctggga
     3421 tggtgaaacc atgaaaaatg gcagcttcag tggattaagt gggggtaatg tggcctgtac
     3481 cctctggttg cataggtatt catacggtta aaatttatca ggcgcgattg cggcagtttt
     3541 tcgggtggtt tgttgccatt tttacctgtc tgctgccgtg atcgcgctga acgcgtttta
     3601 gcggtgcgta caattaaggg attatggtaa atccacttac tgtctgccct cgtagccatc
     3661 gagataaacc gcagtactcc ggccacgatg cgtccggcgt agaggatcga gatcttcaat
     3721 attggccatt agccatatta ttcattggtt atatagcata aatcaatatt ggctattggc
     3781 cattgcatac gttgtatcta tatcataata tgtacattta tattggctca tgtccaatat
     3841 gaccgccatg ttggcattga ttattgacta gttattaata gtaatcaatt acggggtcat
     3901 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     3961 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     4021 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     4081 tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta
     4141 aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt
     4201 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg
     4261 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     4321 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc
     4381 cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt
     4441 tagtgaaccg tcagatcact agaagcttta ttgcggtagt ttatcacagt taaattgcta
     4501 acgcagtcag tgcttctgac acaacagtct cgaacttaag ctgcagaagt tggtcgtgag
     4561 gcactgggca ggtaagtatc aaggttacaa gacaggttta aggagaccaa tagaaactgg
     4621 gcttgtcgag acagagaaga ctcttgcgtt tctgataggc acctattggt cttactgaca
     4681 tccactttgc ctttctctcc acaggtgtcc actcccagtt caattacagc tcttaaggct
     4741 agagtattaa tacgactcac tatagggcta gcaaagccac catggcagaa atcggtactg
     4801 gctttccatt cgacccccat tatgtggaag tcctgggcga gcgcatgcac tacgtcgatg
     4861 ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg taacccgacc tcctcctacg
     4921 tgtggcgcaa catcatcccg catgttgcac cgacccatcg ctgcattgct ccagacctga
     4981 tcggtatggg caaatccgac aaaccagacc tgggttattt cttcgacgac cacgtccgct
     5041 tcatggatgc cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc attcacgact
     5101 ggggctccgc tctgggtttc cactgggcca agcgcaatcc agagcgcgtc aaaggtattg
     5161 catttatgga gttcatccgc cctatcccga cctgggacga atggccagaa tttgcccgcg
     5221 agaccttcca ggccttccgc accaccgacg tcggccgcaa gctgatcatc gatcagaacg
     5281 tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa gtcgagatgg
     5341 accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg cgcttcccaa
     5401 acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa gaatacatgg
     5461 actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca ggcgttctga
     5521 tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag gctgtggaca
     5581 tcggcccggg tctgaatctg ctgcaagaag acaacccgga cctgatcggc agcgagatcg
     5641 cgcgctggct gtcgacgctc gagatttccg gcgagccaac cactgaggat ctgtactttc
     5701 agagcgataa cgcgatcgct tccgaattcg gatccatgaa cctggcgagc cagagcgggg
     5761 aggccggcgc cggccagctg ctcttcgcca acttcaacca ggacaacacg tccctagctg
     5821 ttggtagtaa gtccggttat aaatttttct ccctttcttc tgtggataag ctggaacaga
     5881 tctatgaatg caccgatacg gaagatgtgt gcattgtaga gagattgttc tccagcagcg
     5941 aagtggccat cgtgagcctt aaagcaccaa ggaagctaaa ggtttgccac tttaagaagg
     6001 gaactgagat ctgcaactac agctactcca acacgattct ggctgtgaag ctcaacaggc
     6061 agaggctgat agtatgcctg gaggagtccc tgtacatcca caacattcgg gacatgaagg
     6121 tgctgcatac gatcagggag acgcctccaa accctgcagg cctgtgtgcg ctgtcaatca
     6181 acaacgacaa ctgctacttg gcgtacccag ggagcgcgac catcggagag gtgcaggtct
     6241 tcgataccat taatttgaga gctgcaaaca tgattccggc tcacgacagt cctttagcgg
     6301 cactggcctt tgacgcaagt ggaactaaac ttgccacggc ttcggagaag gggaccgtga
     6361 ttagggtatt ttccattcca gaaggacaaa aactctttga gtttcggaga ggagtaaaga
     6421 ggtgcgtgag catctgctcc ctggccttca gcatggacgg catgttcctc tccgcctcca
     6481 gcaacactga gaccgtgcac atcttcaaac tcgagactgt gaaagaaaaa cccccagagg
     6541 agcccaccac ctggaccggg tacttcggga aagtgctcat ggcctccacc agctacctgc
     6601 cttcccaagt gacagaaatg ttcaaccagg gcagagcctt cgccacggtc cgcctgccat
     6661 tctgcggcca caaaaacatc tgctcgctag ccacaattca gaagatcccg cggttgttgg
     6721 tgggtgccgc cgacgggtac ctgtacatgt acaacctgga cccccaggag ggcggcgagt
     6781 gtgccctgat gaagcagcac cggctggacg gcagtctgga aacgaccaat gagatcttgg
     6841 actctgcctc tcacgactgc cccttagtca ctcagacata cggcgcagct gcaggaaaag
     6901 gtacttacgt gccttcatcc ccaacgagac ttgcctacac agacgacctg ggtgctgtgg
     6961 gtggcgcctg cctggaggac gaggccagcg ccctgcgcct ggatgaggac agcgagcacc
     7021 cgcccatgat tcttcggact gactgagcgg ccgcttgctg agttggctgc tgccaccgct
     7081 gagcaataac tagcataacc ccttggccgc ttcgagcaga catgataaga tacattgatg
     7141 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     7201 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     7261 gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttta agcaagtaaa
     7321 acctctacaa atgtggtaaa atcgaatttt aacaaaatat taacgcttac aatttcctga
     7381 tgcggtattt tctccttacg catctgtgcg
//
