LOCUS       sequence-354965-        6416 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression of human WIPI2d with N-terminal mCherry and 
            C-terminal Strep.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6416)
  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 6416)
  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-mcherry-WIPI2d-cs-TEV-STREP
FEATURES             Location/Qualifiers
     source          1..6416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     496..518
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     580..599
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      646..655
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             652..1359
                     /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(797..815)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1065..1084
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2677..2697
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     regulatory      2702..2711
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2713..2736
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2773..2796
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(2914..2933)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2979..3034
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3033..3052)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3395..3411)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3395..3411)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3408..3430)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3419..3435
                     /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(3443..3449,3450..3467,3468..3473))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3488..3509
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3568..3763
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3614..3749
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3769..3903
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3806..3825)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3860..3879
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3971..3988)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4142..4730)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4222..4241)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4901..5692,5693..5761))
                     /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     5524..5543
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5762..5866)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5897..6276
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg
       61 gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg
      121 gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct
      181 cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca
      241 gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct
      301 gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc
      361 ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc
      421 gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg
      481 gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta
      541 accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc
      601 tgtctcatca ttttggcaaa gaattcctcg atcgacggta tcgatgccac catggtgagc
      661 aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg
      721 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
      781 gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
      841 gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac
      901 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
      961 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc
     1021 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     1081 aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag
     1141 ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag
     1201 accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag
     1261 ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag
     1321 ggccgccact ccaccggcgg catggacgag ctgtacaagt ccggactcag atctcgagct
     1381 caagcttcga attcgatgaa cctggcgagc cagagcgggg aggccggcgc cggccagctg
     1441 ctcttcgcca acttcaacca ggacaacacg tccctagctg ttggtagtaa gtccggttat
     1501 aaatttttct ccctttcttc tgtggataag ctggaacaga tctatgaatg caccgatacg
     1561 gaagatgtgt gcattgtaga gagattgttc tccagcagcc tagtggccat cgtgagcctt
     1621 aaagcaccaa ggaagctaaa ggtttgccac tttaagaagg gaactgagat ctgcaactac
     1681 agctactcca acacgattct ggctgtgaag ctcaacaggc agaggctgat agtatgcctg
     1741 gaggagtccc tgtacatcca caacattcgg gacatgaagg tgctgcatac gatcagggag
     1801 acgcctccaa accctgcagg cctgtgtgcg ctgtcaatca acaacgacaa ctgctacttg
     1861 gcgtacccag ggagcgcgac catcggagag gtgcaggtct tcgataccat taatttgaga
     1921 gctgcaaaca tgattccggc tcacgacagt cctttagcgg cactggcctt tgacgcaagt
     1981 ggaactaaac ttgccacggc ttcggagaag gggaccgtga ttagggtatt ttccattcca
     2041 gaaggacaaa aactctttga gtttcggaga ggagtaaaga ggtgcgtgag catctgctcc
     2101 ctggccttca gcatggacgg catgttcctc tccgcctcca gcaacactga gaccgtgcac
     2161 atcttcaaac tcgagactgt gaaagaaaaa cccccagagg agcccaccac ctggaccggg
     2221 tacttcggga aagtgctcat ggcctccacc agctacctgc cttcccaagt gacagaaatg
     2281 ttcaaccagg gcagagcctt cgccacggtc cgcctgccat tctgcggcca caaaaacatc
     2341 tgctcgctag ccacaattca gaagatcccg cggttgttgg tgggtgccgc cgacgggtac
     2401 ctgtacatgt acaacctgga cccccaggag ggcggcgagt gtgccctgat gaagcagcac
     2461 cggctggacg gcagtctgga aacgaccaat gagatcttgg actctgcctc tcacgactgc
     2521 cccttagtca ctcagacata cggcgcagct gcaggaaaag cctacacaga cgacctgggt
     2581 gctgtgggtg gcgcctgcct ggaggacgag gccagcgccc tgcgcctgga tgaggacagc
     2641 gagcacccgc ccatgattct tcggactgac ggatcggaaa acctgtattt tcagggtggt
     2701 accatggcgg cctggtcaca cccgcagttc gagaaaggtg gaggtgcccg aggtggatcg
     2761 ggaggtggat cgtggagcca cccgcagttc gaaaaatgac ccgggtgata tcttaattaa
     2821 ttaactcgag gaattcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt
     2881 ggccaatgcc ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg
     2941 ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat
     3001 tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa
     3061 tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct
     3121 ggctgccatg aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc
     3181 tgtccattcc ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg
     3241 ttttgtgtta tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag
     3301 atttttcctc ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc
     3361 cctcgacctg cagcccaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     3421 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     3481 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     3541 cgggaaacct gtcgtgccag cggatccgca tctcaattag tcagcaacca tagtcccgcc
     3601 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3661 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     3721 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctaa cttgtttatt
     3781 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3841 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     3901 atccgctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3961 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4021 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4081 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4141 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4201 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4261 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4321 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4381 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4441 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4501 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4561 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4621 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4681 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4741 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4801 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4861 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4921 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4981 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5041 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5101 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5161 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5221 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5281 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5341 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5401 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5461 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5521 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5581 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5641 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5701 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5761 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5821 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5881 aagtgccacc tgggtcgaca ttgattattg actagttatt aatagtaatc aattacgggg
     5941 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     6001 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     6061 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     6121 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     6181 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     6241 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
     6301 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
     6361 ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg cggggc
//
