LOCUS       sequence-340763-        6256 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression of human WIPI2d H85E with C-terminal Strep.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6256)
  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 6256)
  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: pCAG-WIPI2dH85E-cs-TEV-STREP
FEATURES             Location/Qualifiers
     source          1..6256
                     /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        1269..1648
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1928..2944
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2868..2890
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2952..2971
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             4305..4325
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     regulatory      4330..4339
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4341..4364
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             4401..4424
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(4542..4561)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4607..4662
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4661..4680)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5023..5039)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5023..5039)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5036..5058)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5047..5063
                     /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(5071..5077,5078..5095,5096..5101))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5116..5137
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5196..5391
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5242..5377
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5397..5531
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5434..5453)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5488..5507
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5599..5616)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5770..6256,1..102))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5850..5869)
                     /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 cctgggtcga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     1321 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     1381 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     1441 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     1501 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     1561 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     1621 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
     1681 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
     1741 tgcagcgatg ggggcggggg gggggggggg gsgcgcscsr ggsggggsgg ggsggggsgr
     1801 ggggsggggs ggggsgrggs ggrgrggkgs ggcggcagcc aatcagagcg gcgcgctccg
     1861 aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg
     1921 gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg
     1981 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc
     2041 ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct
     2101 gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg
     2161 tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg
     2221 tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc
     2281 ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg
     2341 gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc
     2401 cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac
     2461 ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggkgg gggtgccggg
     2521 cggggcgggg ccgcctcggg ccggggrggg ctcgggggag gggcgcggcg gcccccggag
     2581 cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga
     2641 gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg
     2701 ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg
     2761 cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg
     2821 ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg
     2881 gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct
     2941 acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattcct
     3001 cgatcgacgg tatcgatgcc accatgaacc tggcgagcca gagcggggag gccggcgccg
     3061 gccagctgct cttcgccaac ttcaaccagg acaacacgtc cctagctgtt ggtagtaagt
     3121 ccggttataa atttttctcc ctttcttctg tggataagct ggaacagatc tatgaatgca
     3181 ccgatacgga agatgtgtgc attgtagaga gattgttctc cagcagccta gtggccatcg
     3241 tgagccttaa agcaccaagg aagctaaagg tttgcgaatt taagaaggga actgagatct
     3301 gcaactacag ctactccaac acgattctgg ctgtgaagct caacaggcag aggctgatag
     3361 tatgcctgga ggagtccctg tacatccaca acattcggga catgaaggtg ctgcatacga
     3421 tcagggagac gcctccaaac cctgcaggcc tgtgtgcgct gtcaatcaac aacgacaact
     3481 gctacttggc gtacccaggg agcgcgacca tcggagaggt gcaggtcttc gataccatta
     3541 atttgagagc tgcaaacatg attccggctc acgacagtcc tttagcggca ctggcctttg
     3601 acgcaagtgg aactaaactt gccacggctt cggagaaggg gaccgtgatt agggtatttt
     3661 ccattccaga aggacaaaaa ctctttgagt ttcggagagg agtaaagagg tgcgtgagca
     3721 tctgctccct ggccttcagc atggacggca tgttcctctc cgcctccagc aacactgaga
     3781 ccgtgcacat cttcaaactc gagactgtga aagaaaaacc cccagaggag cccaccacct
     3841 ggaccgggta cttcgggaaa gtgctcatgg cctccaccag ctacctgcct tcccaagtga
     3901 cagaaatgtt caaccagggc agagccttcg ccacggtccg cctgccattc tgcggccaca
     3961 aaaacatctg ctcgctagcc acaattcaga agatcccgcg gttgttggtg ggtgccgccg
     4021 acgggtacct gtacatgtac aacctggacc cccaggaggg cggcgagtgt gccctgatga
     4081 agcagcaccg gctggacggc agtctggaaa cgaccaatga gatcttggac tctgcctctc
     4141 acgactgccc cttagtcact cagacatacg gcgcagctgc aggaaaagcc tacacagacg
     4201 acctgggtgc tgtgggtggc gcctgcctgg aggacgaggc cagcgccctg cgcctggatg
     4261 aggacagcga gcacccgccc atgattcttc ggactgacgg atcggaaaac ctgtattttc
     4321 agggtggtac catggcggcc tggtcacacc cgcagttcga gaaaggtgga ggtgcccgag
     4381 gtggatcggg aggtggatcg tggagccacc cgcagttcga aaaatgaccc gggtgatatc
     4441 ttaattaatt aactcgagga attcactcct caggtgcagg ctgcctatca gaaggtggtg
     4501 gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa
     4561 aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt
     4621 attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg
     4681 agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc
     4741 catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag
     4801 ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta gatttttttt
     4861 atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta
     4921 ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta
     4981 tggagatccc tcgacctgca gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt
     5041 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5101 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5161 tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata
     5221 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     5281 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     5341 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact
     5401 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5461 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5521 atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5581 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5641 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5701 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5761 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5821 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5881 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5941 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6001 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6061 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6121 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6181 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6241 gccagttacc ttcgga
//
