LOCUS       sequence-350264-        7410 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses WIPI2B (H85E K88E) 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 Apr 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
FEATURES             Location/Qualifiers
     source          1..7410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          720..852
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     835..854
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     896..915
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        896..914
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      924..933
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             930..1820
                     /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             1833..1853
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(3224..3242)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3274..3395
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3285..3304)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3339..3358
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3640..3997
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3640..3831
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3663..3683)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3848..3983
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4048..4842
                     /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(4102..4121)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4712..4731
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4906..4954
                     /note="synthetic polyadenylation signal"
     promoter        5110..5214
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5215..5283,5284..6075)
                     /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(5433..5452)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6233..6821)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6313..6332)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6937..7220
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatcactag aagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg
      661 cttctgacac aacagtctcg aacttaagct gcagaagttg gtcgtgaggc actgggcagg
      721 taagtatcaa ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac
      781 agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct
      841 ttctctccac aggtgtccac tcccagttca attacagctc ttaaggctag agtattaata
      901 cgactcacta tagggctagc aaagccacca tggcagaaat cggtactggc tttccattcg
      961 acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg
     1021 atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca
     1081 tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca
     1141 aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct
     1201 tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc
     1261 tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt
     1321 tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag accttccagg
     1381 ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg
     1441 gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg
     1501 agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa
     1561 tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc
     1621 agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg
     1681 aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc
     1741 tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt
     1801 cgacgctcga gatttccggc gagccaacca ctgaggatct gtactttcag agcgataacg
     1861 cgatcgcttc cgaattcgga tccatgaacc tggcgagcca gagcggggag gccggcgccg
     1921 gccagctgct cttcgccaac ttcaaccagg acaacacgtc cctagctgtt ggtagtaagt
     1981 ccggttataa atttttctcc ctttcttctg tggataagct ggaacagatc tatgaatgca
     2041 ccgatacgga agatgtgtgc attgtagaga gattgttctc cagcagccta gtggccatcg
     2101 tgagccttaa agcaccaagg aagctaaagg tttgcgagtt taaggaggga actgagatct
     2161 gcaactacag ctactccaac acgattctgg ctgtgaagct caacaggcag aggctgatag
     2221 tatgcctgga ggagtccctg tacatccaca acattcggga catgaaggtg ctgcatacga
     2281 tcagggagac gcctccaaac cctgcaggcc tgtgtgcgct gtcaatcaac aacgacaact
     2341 gctacttggc gtacccaggg agcgcgacca tcggagaggt gcaggtcttc gataccatta
     2401 atttgagagc tgcaaacatg attccggctc acgacagtcc tttagcggca ctggcctttg
     2461 acgcaagtgg aactaaactt gccacggctt cggagaaggg gaccgtgatt agggtatttt
     2521 ccattccaga aggacaaaaa ctctttgagt ttcggagagg agtaaagagg tgcgtgagca
     2581 tctgctccct ggccttcagc atggacggca tgttcctctc cgcctccagc aacactgaga
     2641 ccgtgcacat cttcaaactc gagactgtga aagaaaaacc cccagaggag cccaccacct
     2701 ggaccgggta cttcgggaaa gtgctcatgg cctccaccag ctacctgcct tcccaagtga
     2761 cagaaatgtt caaccagggc agagccttcg ccacggtccg cctgccattc tgcggccaca
     2821 aaaacatctg ctcgctagcc acaattcaga agatcccgcg gttgttggtg ggtgccgccg
     2881 acgggtacct gtacatgtac aacctggacc cccaggaggg cggcgagtgt gccctgatga
     2941 agcagcaccg gctggacggc agtctggaaa cgaccaatga gatcttggac tctgcctctc
     3001 acgactgccc cttagtcact cagacatacg gcgcagctgc aggaaaaggt acttacgtgc
     3061 cttcatcccc aacgagactt gcctacacag acgacctggg tgctgtgggt ggcgcctgcc
     3121 tggaggacga ggccagcgcc ctgcgcctgg atgaggacag cgagcacccg cccatgattc
     3181 ttcggactga ctgagcggcc gcttgctgag ttggctgctg ccaccgctga gcaataacta
     3241 gcataacccc ttggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa
     3301 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     3361 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     3421 tgtttcaggt tcagggggag atgtgggagg tttttttaag caagtaaaac ctctacaaat
     3481 gtggtaaaat cgaattttaa caaaatatta acgcttacaa tttcctgatg cggtattttc
     3541 tccttacgca tctgtgcggt atttcacacc gcatacgcgg atctgcgcag caccatggcc
     3601 tgaaataacc tctgaaagag gaacttggtt aggtaccttc tgaggcggaa agaaccagct
     3661 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3721 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     3781 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3841 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3901 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     3961 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag cttgattctt ctgacacaac
     4021 agtctcgaac ttaaggctag agccaccatg attgaacaag atggattgca cgcaggttct
     4081 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4141 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4201 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4261 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4321 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4381 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4441 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4501 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4561 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4621 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4681 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4741 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4801 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     4861 aaatgaccga ccaagcgacg cccaacctgc catcacgatg gccgcaataa aatatcttta
     4921 ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagcgataag gatcctcttt
     4981 gcgcttgcgt tttcccttgt ccagatagcc cagtagctga cattcatccg gggtcagcac
     5041 cgtttctgcg gactggcttt ctacgtaatg gtttcttaga cgtcaggtgg cacttttcgg
     5101 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5161 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5221 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5281 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5341 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5401 cgttttccaa tgatgagcac tttcaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5461 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5521 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5581 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac tatcggagga
     5641 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5701 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5761 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5821 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5881 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     5941 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6001 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6061 attaagcatt ggtaattcga aatgaccgac caagcgacgc ccaaccggta tcagctcact
     6121 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6181 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6241 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6301 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6361 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6421 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6481 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6541 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6601 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6661 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6721 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6781 tttgcaagca gcagattacg cgcagaaaaa aaggatttca agaagatcct ttgatctttt
     6841 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6901 tatcaaaaag gatcttcacc tagatccttt tatagtccgg aaatacagga acgcacgctg
     6961 gatggccctt cgctgggatg gtgaaaccat gaaaaatggc agcttcagtg gattaagtgg
     7021 gggtaatgtg gcctgtaccc tctggttgca taggtattca tacggttaaa atttatcagg
     7081 cgcgattgcg gcagtttttc gggtggtttg ttgccatttt tacctgtctg ctgccgtgat
     7141 cgcgctgaac gcgttttagc ggtgcgtaca attaagggat tatggtaaat ccacttactg
     7201 tctgccctcg tagccatcga gataaaccgc agtactccgg ccacgatgcg tccggcgtag
     7261 aggatcgaga tcttcaatat tggccattag ccatattatt cattggttat atagcataaa
     7321 tcaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg tacatttata
     7381 ttggctcatg tccaatatga ccgccatgtt
//
