LOCUS       sequence-337619-        7009 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the purifcation of GFP- WIPI2d protein .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7009)
  AUTHORS   Chang C, Shi X, Jensen LE, Yokom AL, Fracchiolla D, Martens S, 
            Hurley JH
  TITLE     Reconstitution of cargo-induced LC3 lipidation in mammalian 
            selective autophagy.
  JOURNAL   Sci Adv. 2021 Apr 23;7(17). pii: 7/17/eabg4922. doi: 
            10.1126/sciadv.abg4922. Print 2021 Apr.
  PUBMED    33893090
REFERENCE   2  (bases 1 to 7009)
  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: "Sci Adv."; 
            date: "2021-04-23"; volume: "7(17). pii: 7/17/eabg4922. doi"; pages:
            " 10.1126/sciadv.abg4922. Print 2021 Apr"
COMMENT     Sequence Label: GFP-WIPI2d-TEVcs-TSF
FEATURES             Location/Qualifiers
     source          1..7009
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..663
                     /label=chicken beta-actin promoter
     intron          664..1680
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1604..1626
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1688..1707
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1754..1763
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1760..1762,1763..1765,1766..2476)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1805..1826)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2066..2085)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2413..2434
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3794..3814
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     regulatory      3819..3828
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3830..3853
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3890..3913
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(4031..4050)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4096..4151
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4150..4169)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4512..4528)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4512..4528)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4525..4547)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4536..4552
                     /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(4560..4566,4567..4584,4585..4590))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4605..4626
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4685..4880
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4731..4866
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4886..5020
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4923..4942)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4977..4996
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5088..5105)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5259..5847)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5339..5358)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6018..6809,6810..6878))
                     /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     6641..6660
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6879..6983)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggggcgc gcgccrgggg gggggggggg gggggrgggg
      541 gggggsgggg sgrggcggag aggkgcggcg gcagccaatc agagcggcgc gctccgaaag
      601 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
      661 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
      721 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
      781 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
      841 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag
      961 cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc
     1021 gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt
     1081 gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg
     1141 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg
     1201 cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg
     1261 gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc
     1321 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg
     1381 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc
     1441 accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg
     1501 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt
     1561 ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt
     1621 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     1681 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcctcgat
     1741 cgacggtatc gatgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1801 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1861 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1921 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     1981 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2041 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2101 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2161 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2221 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2281 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     2341 gctgctgccc gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga
     2401 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     2461 ggacgagctg tacaagtccg gactcagatc tcgagctcaa gcttcgaatt cgatgaacct
     2521 ggcgagccag agcggggagg ccggcgccgg ccagctgctc ttcgccaact tcaaccagga
     2581 caacacgtcc ctagctgttg gtagtaagtc cggttataaa tttttctccc tttcttctgt
     2641 ggataagctg gaacagatct atgaatgcac cgatacggaa gatgtgtgca ttgtagagag
     2701 attgttctcc agcagcctag tggccatcgt gagccttaaa gcaccaagga agctaaaggt
     2761 ttgccacttt aagaagggaa ctgagatctg caactacagc tactccaaca cgattctggc
     2821 tgtgaagctc aacaggcaga ggctgatagt atgcctggag gagtccctgt acatccacaa
     2881 cattcgggac atgaaggtgc tgcatacgat cagggagacg cctccaaacc ctgcaggcct
     2941 gtgtgcgctg tcaatcaaca acgacaactg ctacttggcg tacccaggga gcgcgaccat
     3001 cggagaggtg caggtcttcg ataccattaa tttgagagct gcaaacatga ttccggctca
     3061 cgacagtcct ttagcggcac tggcctttga cgcaagtgga actaaacttg ccacggcttc
     3121 ggagaagggg accgtgatta gggtattttc cattccagaa ggacaaaaac tctttgagtt
     3181 tcggagagga gtaaagaggt gcgtgagcat ctgctccctg gccttcagca tggacggcat
     3241 gttcctctcc gcctccagca acactgagac cgtgcacatc ttcaaactcg agactgtgaa
     3301 agaaaaaccc ccagaggagc ccaccacctg gaccgggtac ttcgggaaag tgctcatggc
     3361 ctccaccagc tacctgcctt cccaagtgac agaaatgttc aaccagggca gagccttcgc
     3421 cacggtccgc ctgccattct gcggccacaa aaacatctgc tcgctagcca caattcagaa
     3481 gatcccgcgg ttgttggtgg gtgccgccga cgggtacctg tacatgtaca acctggaccc
     3541 ccaggagggc ggcgagtgtg ccctgatgaa gcagcaccgg ctggacggca gtctggaaac
     3601 gaccaatgag atcttggact ctgcctctca cgactgcccc ttagtcactc agacatacgg
     3661 cgcagctgca ggaaaagcct acacagacga cctgggtgct gtgggtggcg cctgcctgga
     3721 ggacgaggcc agcgccctgc gcctggatga ggacagcgag cacccgccca tgattcttcg
     3781 gactgacgga tcggaaaacc tgtattttca gggtggtacc atggcggcct ggtcacaccc
     3841 gcagttcgag aaaggtggag gtgcccgagg tggatcggga ggtggatcgt ggagccaccc
     3901 gcagttcgaa aaatgacccg ggtgatatct taattaatta actcgaggaa ttcactcctc
     3961 aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat
     4021 accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc
     4081 atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt
     4141 tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt
     4201 atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc
     4261 tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa
     4321 aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa
     4381 catccctaaa attttcctta catgttttac tagccagatt tttcctcctc tcctgactac
     4441 tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag cccaagcttg
     4501 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4561 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4621 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg
     4681 atccgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4741 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4801 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4861 gaggcctagg cttttgcaaa aagctaactt gtttattgca gcttataatg gttacaaata
     4921 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4981 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cgctgcatta atgaatcggc
     5041 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5101 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5161 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5221 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5281 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5341 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5401 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5461 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5521 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5581 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5641 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5701 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5761 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5821 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5881 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5941 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6001 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6061 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6121 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6181 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6241 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6301 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6361 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6421 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6481 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6541 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6601 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6661 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6721 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6781 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6841 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6901 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6961 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctg
//
