LOCUS       sequence-350261-        7410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses WIPI2B (H85E K88E I92E) 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 H85E K88E I92E
FEATURES             Location/Qualifiers
     source          1..7410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          340..472
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     455..474
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     516..535
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        516..534
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      544..553
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             550..1440
                     /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             1453..1473
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(2844..2862)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    2894..3015
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2905..2924)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2959..2978
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3260..3617
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3260..3451
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3283..3303)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3468..3603
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3668..4462
                     /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(3722..3741)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4332..4351
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4526..4574
                     /note="synthetic polyadenylation signal"
     promoter        4730..4834
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4835..4903,4904..5695)
                     /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(5053..5072)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5853..6441)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5933..5952)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6557..6840
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     enhancer        7031..7410
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatcactag aagctttatt
      241 gcggtagttt atcacagtta aattgctaac gcagtcagtg cttctgacac aacagtctcg
      301 aacttaagct gcagaagttg gtcgtgaggc actgggcagg taagtatcaa ggttacaaga
      361 caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact cttgcgtttc
      421 tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac aggtgtccac
      481 tcccagttca attacagctc ttaaggctag agtattaata cgactcacta tagggctagc
      541 aaagccacca tggcagaaat cggtactggc tttccattcg acccccatta tgtggaagtc
      601 ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc
      661 ctgcacggta acccgacctc ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg
      721 acccatcgct gcattgctcc agacctgatc ggtatgggca aatccgacaa accagacctg
      781 ggttatttct tcgacgacca cgtccgcttc atggatgcct tcatcgaagc cctgggtctg
      841 gaagaggtcg tcctggtcat tcacgactgg ggctccgctc tgggtttcca ctgggccaag
      901 cgcaatccag agcgcgtcaa aggtattgca tttatggagt tcatccgccc tatcccgacc
      961 tgggacgaat ggccagaatt tgcccgcgag accttccagg ccttccgcac caccgacgtc
     1021 ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc gatgggtgtc
     1081 gtccgcccgc tgactgaagt cgagatggac cattaccgcg agccgttcct gaatcctgtt
     1141 gaccgcgagc cactgtggcg cttcccaaac gagctgccaa tcgccggtga gccagcgaac
     1201 atcgtcgcgc tggtcgaaga atacatggac tggctgcacc agtcccctgt cccgaagctg
     1261 ctgttctggg gcaccccagg cgttctgatc ccaccggccg aagccgctcg cctggccaaa
     1321 agcctgccta actgcaaggc tgtggacatc ggcccgggtc tgaatctgct gcaagaagac
     1381 aacccggacc tgatcggcag cgagatcgcg cgctggctgt cgacgctcga gatttccggc
     1441 gagccaacca ctgaggatct gtactttcag agcgataacg cgatcgcttc cgaattcgga
     1501 tccatgaacc tggcgagcca gagcggggag gccggcgccg gccagctgct cttcgccaac
     1561 ttcaaccagg acaacacgtc cctagctgtt ggtagtaagt ccggttataa atttttctcc
     1621 ctttcttctg tggataagct ggaacagatc tatgaatgca ccgatacgga agatgtgtgc
     1681 attgtagaga gattgttctc cagcagccta gtggccatcg tgagccttaa agcaccaagg
     1741 aagctaaagg tttgcgagtt taaggaggga actgaggaat gcaactacag ctactccaac
     1801 acgattctgg ctgtgaagct caacaggcag aggctgatag tatgcctgga ggagtccctg
     1861 tacatccaca acattcggga catgaaggtg ctgcatacga tcagggagac gcctccaaac
     1921 cctgcaggcc tgtgtgcgct gtcaatcaac aacgacaact gctacttggc gtacccaggg
     1981 agcgcgacca tcggagaggt gcaggtcttc gataccatta atttgagagc tgcaaacatg
     2041 attccggctc acgacagtcc tttagcggca ctggcctttg acgcaagtgg aactaaactt
     2101 gccacggctt cggagaaggg gaccgtgatt agggtatttt ccattccaga aggacaaaaa
     2161 ctctttgagt ttcggagagg agtaaagagg tgcgtgagca tctgctccct ggccttcagc
     2221 atggacggca tgttcctctc cgcctccagc aacactgaga ccgtgcacat cttcaaactc
     2281 gagactgtga aagaaaaacc cccagaggag cccaccacct ggaccgggta cttcgggaaa
     2341 gtgctcatgg cctccaccag ctacctgcct tcccaagtga cagaaatgtt caaccagggc
     2401 agagccttcg ccacggtccg cctgccattc tgcggccaca aaaacatctg ctcgctagcc
     2461 acaattcaga agatcccgcg gttgttggtg ggtgccgccg acgggtacct gtacatgtac
     2521 aacctggacc cccaggaggg cggcgagtgt gccctgatga agcagcaccg gctggacggc
     2581 agtctggaaa cgaccaatga gatcttggac tctgcctctc acgactgccc cttagtcact
     2641 cagacatacg gcgcagctgc aggaaaaggt acttacgtgc cttcatcccc aacgagactt
     2701 gcctacacag acgacctggg tgctgtgggt ggcgcctgcc tggaggacga ggccagcgcc
     2761 ctgcgcctgg atgaggacag cgagcacccg cccatgattc ttcggactga ctgagcggcc
     2821 gcttgctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggccgctt
     2881 cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     2941 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     3001 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     3061 atgtgggagg tttttttaag caagtaaaac ctctacaaat gtggtaaaat cgaattttaa
     3121 caaaatatta acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt
     3181 atttcacacc gcatacgcgg atctgcgcag caccatggcc tgaaataacc tctgaaagag
     3241 gaacttggtt aggtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     3301 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3361 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3421 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3481 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3541 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3601 gcctaggctt ttgcaaaaag cttgattctt ctgacacaac agtctcgaac ttaaggctag
     3661 agccaccatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     3721 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     3781 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     3841 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     3901 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     3961 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     4021 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     4081 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     4141 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     4201 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     4261 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     4321 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     4381 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     4441 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     4501 cccaacctgc catcacgatg gccgcaataa aatatcttta ttttcattac atctgtgtgt
     4561 tggttttttg tgtgaatcga tagcgataag gatcctcttt gcgcttgcgt tttcccttgt
     4621 ccagatagcc cagtagctga cattcatccg gggtcagcac cgtttctgcg gactggcttt
     4681 ctacgtaatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4741 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4801 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     4861 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     4921 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     4981 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5041 tttcaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5101 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5161 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5221 taacactgcg gccaacttac ttctgacaac tatcggagga ccgaaggagc taaccgcttt
     5281 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5341 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     5401 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5461 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5521 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     5581 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     5641 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaattcga
     5701 aatgaccgac caagcgacgc ccaaccggta tcagctcact caaaggcggt aatacggtta
     5761 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5821 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5881 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5941 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     6001 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     6061 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6121 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6181 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6241 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     6301 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6361 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6421 cgcagaaaaa aaggatttca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6481 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6541 tagatccttt tatagtccgg aaatacagga acgcacgctg gatggccctt cgctgggatg
     6601 gtgaaaccat gaaaaatggc agcttcagtg gattaagtgg gggtaatgtg gcctgtaccc
     6661 tctggttgca taggtattca tacggttaaa atttatcagg cgcgattgcg gcagtttttc
     6721 gggtggtttg ttgccatttt tacctgtctg ctgccgtgat cgcgctgaac gcgttttagc
     6781 ggtgcgtaca attaagggat tatggtaaat ccacttactg tctgccctcg tagccatcga
     6841 gataaaccgc agtactccgg ccacgatgcg tccggcgtag aggatcgaga tcttcaatat
     6901 tggccattag ccatattatt cattggttat atagcataaa tcaatattgg ctattggcca
     6961 ttgcatacgt tgtatctata tcataatatg tacatttata ttggctcatg tccaatatga
     7021 ccgccatgtt ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     7081 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     7141 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     7201 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     7261 gcagtacatc aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa
     7321 tggcccgcct ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac
     7381 atctacgtat tagtcatcgc tattaccatg
//
