LOCUS       sequence-340915-        7000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human WIPI2d K128E with N-terminal mCherry 
            and C-terminal Strep.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7000)
  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 7000)
  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-mcherry-WIPI2dK128E-cs-TEV-STREP
FEATURES             Location/Qualifiers
     source          1..7000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     73..92
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      139..148
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             145..852
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(290..308)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     558..577
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2170..2190
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     regulatory      2195..2204
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2206..2229
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2266..2289
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(2407..2426)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2472..2527
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2526..2545)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2888..2904)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2888..2904)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2901..2923)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2912..2928
                     /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(2936..2942,2943..2960,2961..2966))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2981..3002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3061..3256
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3107..3242
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3262..3396
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3299..3318)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3353..3372
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3464..3481)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3635..4223)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3715..3734)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4394..5185,5186..5254))
                     /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     5017..5036
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5255..5359)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5390..5769
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          join(6049..7000,1..65)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     join(6989..7000,1..11)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
ORIGIN
        1 ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc
       61 tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattcc
      121 tcgatcgacg gtatcgatgc caccatggtg agcaagggcg aggaggataa catggccatc
      181 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc
      241 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
      301 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
      361 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
      421 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
      481 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
      541 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc
      601 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg
      661 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg
      721 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac
      781 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac
      841 gagctgtaca agtccggact cagatctcga gctcaagctt cgaattcgat gaacctggcg
      901 agccagagcg gggaggccgg cgccggccag ctgctcttcg ccaacttcaa ccaggacaac
      961 acgtccctag ctgttggtag taagtccggt tataaatttt tctccctttc ttctgtggat
     1021 aagctggaac agatctatga atgcaccgat acggaagatg tgtgcattgt agagagattg
     1081 ttctccagca gcctagtggc catcgtgagc cttaaagcac caaggaagct aaaggtttgc
     1141 cactttaaga agggaactga gatctgcaac tacagctact ccaacacgat tctggctgtg
     1201 aagctcaaca ggcagaggct gatagtatgc ctggaggagt ccctgtacat ccacaacatt
     1261 cgggacatgg aggtgctgca tacgatcagg gagacgcctc caaaccctgc aggcctgtgt
     1321 gcgctgtcaa tcaacaacga caactgctac ttggcgtacc cagggagcgc gaccatcgga
     1381 gaggtgcagg tcttcgatac cattaatttg agagctgcaa acatgattcc ggctcacgac
     1441 agtcctttag cggcactggc ctttgacgca agtggaacta aacttgccac ggcttcggag
     1501 aaggggaccg tgattagggt attttccatt ccagaaggac aaaaactctt tgagtttcgg
     1561 agaggagtaa agaggtgcgt gagcatctgc tccctggcct tcagcatgga cggcatgttc
     1621 ctctccgcct ccagcaacac tgagaccgtg cacatcttca aactcgagac tgtgaaagaa
     1681 aaacccccag aggagcccac cacctggacc gggtacttcg ggaaagtgct catggcctcc
     1741 accagctacc tgccttccca agtgacagaa atgttcaacc agggcagagc cttcgccacg
     1801 gtccgcctgc cattctgcgg ccacaaaaac atctgctcgc tagccacaat tcagaagatc
     1861 ccgcggttgt tggtgggtgc cgccgacggg tacctgtaca tgtacaacct ggacccccag
     1921 gagggcggcg agtgtgccct gatgaagcag caccggctgg acggcagtct ggaaacgacc
     1981 aatgagatct tggactctgc ctctcacgac tgccccttag tcactcagac atacggcgca
     2041 gctgcaggaa aagcctacac agacgacctg ggtgctgtgg gtggcgcctg cctggaggac
     2101 gaggccagcg ccctgcgcct ggatgaggac agcgagcacc cgcccatgat tcttcggact
     2161 gacggatcgg aaaacctgta ttttcagggt ggtaccatgg cggcctggtc acacccgcag
     2221 ttcgagaaag gtggaggtgc ccgaggtgga tcgggaggtg gatcgtggag ccacccgcag
     2281 ttcgaaaaat gacccgggtg atatcttaat taattaactc gaggaattca ctcctcaggt
     2341 gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca
     2401 ctgagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct
     2461 gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg
     2521 tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt
     2581 ggtttagagt ttggcaacat atgcccatat gctggctgcc atgaacaaag gttggctata
     2641 aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc
     2701 cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc
     2761 cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc
     2821 agtcatagct gtccctcttc tcttatggag atccctcgac ctgcagccca agcttggcgt
     2881 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2941 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     3001 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc
     3061 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3121 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3181 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3241 cctaggcttt tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc
     3301 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     3361 tccaaactca tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac
     3421 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     3481 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3541 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3601 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3661 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3721 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3781 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3841 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3901 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3961 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4021 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4081 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4141 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4201 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4261 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4321 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4381 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4441 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4501 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4561 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4621 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4681 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4741 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4801 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4861 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4921 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4981 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5041 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5101 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5161 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5221 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5281 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5341 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgggtcg acattgatta
     5401 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     5461 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     5521 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     5581 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     5641 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     5701 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     5761 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
     5821 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
     5881 gggggggggg ggsgcgcgcs rggsggggsg gggsggggsg rggggsgggg sggggsgrgg
     5941 sggrgrggkg sggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
     6001 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
     6061 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
     6121 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
     6181 tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg
     6241 ctccgggagg gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc
     6301 gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc
     6361 gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg cggtgccccg
     6421 cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg
     6481 tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga
     6541 gttgctgagc acggcccggc ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc
     6601 gccgtgccgg gcggggggtg gcggcaggkg ggggtgccgg gcggggcggg gccgcctcgg
     6661 gccggggrgg gctcggggga ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc
     6721 gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct
     6781 ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg
     6841 cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg
     6901 tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc
     6961 tgccttcggg ggggacgggg cagggcgggg ttcggcttct
//
