LOCUS       sequence-333211-        6275 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human WIPI2d K88E with C-terminal Strep.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6275)
  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 6275)
  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: pCAG-WIPI2dK88E-cs-TEV-STREP
FEATURES             Location/Qualifiers
     source          1..6275
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(273..1064,1065..1133))
                     /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     896..915
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1134..1238)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1268..1647
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1649..1926
                     /label=chicken beta-actin promoter
     intron          1927..2943
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2867..2889
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2951..2970
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             4304..4324
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     regulatory      4329..4338
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4340..4363
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             4400..4423
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(4541..4560)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4606..4661
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4660..4679)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5022..5038)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5022..5038)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5035..5057)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5046..5062
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5070..5076,5077..5094,5095..5100))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5115..5136
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    5416..5550
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5453..5472)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5507..5526
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5618..5635)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5789..6275,1..102))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5869..5888)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
       61 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
      121 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
      181 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
      241 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
      301 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
      361 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
      421 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
      481 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
      541 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
      601 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
      661 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
      721 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
      781 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
      841 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
      901 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
      961 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     1021 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     1081 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     1141 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     1201 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     1261 cctggtcgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
     1321 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     1381 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     1441 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     1501 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     1561 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     1621 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
     1681 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
     1741 gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag
     1801 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
     1861 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
     1921 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgcgcc gcctcgcgcc
     1981 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
     2041 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc
     2101 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg
     2161 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg
     2221 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg
     2281 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg
     2341 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc
     2401 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg
     2461 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc
     2521 ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc
     2581 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag
     2641 agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc
     2701 cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
     2761 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc
     2821 tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg
     2881 cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta
     2941 cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattcctc
     3001 gatcgacggt atcgatgcca ccatgaacct ggcgagccag agcggggagg ccggcgccgg
     3061 ccagctgctc ttcgccaact tcaaccagga caacacgtcc ctagctgttg gtagtaagtc
     3121 cggttataaa tttttctccc tttcttctgt ggataagctg gaacagatct atgaatgcac
     3181 cgatacggaa gatgtgtgca ttgtagagag attgttctcc agcagcctag tggccatcgt
     3241 gagccttaaa gcaccaagga agctaaaggt ttgccacttt aaggagggaa ctgagatctg
     3301 caactacagc tactccaaca cgattctggc tgtgaagctc aacaggcaga ggctgatagt
     3361 atgcctggag gagtccctgt acatccacaa cattcgggac atgaaggtgc tgcatacgat
     3421 cagggagacg cctccaaacc ctgcaggcct gtgtgcgctg tcaatcaaca acgacaactg
     3481 ctacttggcg tacccaggga gcgcgaccat cggagaggtg caggtcttcg ataccattaa
     3541 tttgagagct gcaaacatga ttccggctca cgacagtcct ttagcggcac tggcctttga
     3601 cgcaagtgga actaaacttg ccacggcttc ggagaagggg accgtgatta gggtattttc
     3661 cattccagaa ggacaaaaac tctttgagtt tcggagagga gtaaagaggt gcgtgagcat
     3721 ctgctccctg gccttcagca tggacggcat gttcctctcc gcctccagca acactgagac
     3781 cgtgcacatc ttcaaactcg agactgtgaa agaaaaaccc ccagaggagc ccaccacctg
     3841 gaccgggtac ttcgggaaag tgctcatggc ctccaccagc tacctgcctt cccaagtgac
     3901 agaaatgttc aaccagggca gagccttcgc cacggtccgc ctgccattct gcggccacaa
     3961 aaacatctgc tcgctagcca caattcagaa gatcccgcgg ttgttggtgg gtgccgccga
     4021 cgggtacctg tacatgtaca acctggaccc ccaggagggc ggcgagtgtg ccctgatgaa
     4081 gcagcaccgg ctggacggca gtctggaaac gaccaatgag atcttggact ctgcctctca
     4141 cgactgcccc ttagtcactc agacatacgg cgcagctgca ggaaaagcct acacagacga
     4201 cctgggtgct gtgggtggcg cctgcctgga ggacgaggcc agcgccctgc gcctggatga
     4261 ggacagcgag cacccgccca tgattcttcg gactgacgga tcggaaaacc tgtattttca
     4321 gggtggtacc atggcggcct ggtcacaccc gcagttcgag aaaggtggag gtgcccgagg
     4381 tggatcggga ggtggatcgt ggagccaccc gcagttcgaa aaatgacccg ggtgatatct
     4441 taattaatta actcgaggaa ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg
     4501 ctggtgtggc caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa
     4561 attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta
     4621 ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga
     4681 gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc
     4741 atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc
     4801 cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta
     4861 tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac
     4921 tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat
     4981 ggagatccct cgacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5041 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5101 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5161 ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca gcaaccatag
     5221 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     5281 cccatggctg actaattttt tttatttatg cagaggccgc ctctacaaat gtggtatgga
     5341 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     5401 cttttgcaaa aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5461 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5521 ctcatcaatg tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg
     5581 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5641 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5701 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5761 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5821 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5881 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5941 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt
     6001 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6061 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6121 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6181 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     6241 gtatctgcgc tctgctgaag ccagttacct tcgga
//
