LOCUS       sequence-337559-        7426 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the purifcation of GFP- PI3KC3 complex .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7426)
  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 7426)
  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: GST-GFP-ATG14
FEATURES             Location/Qualifiers
     source          1..7426
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          203..1219
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1143..1165
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1227..1246
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1295..1304
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1301..1957
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MASPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1915..1937
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1985..2005
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2018..2020,2021..2023,2024..2734)
                     /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(2063..2084)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2324..2343)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2671..2692
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4322..4341)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4387..4442
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4441..4460)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4803..4819)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4803..4819)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4816..4838)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4827..4843
                     /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(4851..4857,4858..4875,4876..4881))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4896..4917
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5046..5063)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5217..5805)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5297..5316)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5976..6767,6768..6836))
                     /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     6599..6618
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6837..6941)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        6972..7351
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggsgcg cgccrggsgg
       61 ggsggggsgg ggsgrggggs ggggsggggs gaggcggaga ggtgcggcgg cagccaatca
      121 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
      181 aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc
      241 cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag
      301 cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt
      361 ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg
      421 gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc
      481 gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt
      541 gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa
      601 caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt
      661 cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg
      721 tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca
      781 ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg
      841 cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt
      901 atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa
      961 atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg
     1021 caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc
     1081 tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc
     1141 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc
     1201 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt
     1261 tggcaaagaa ttgcggccgt ctcaggccga attcgccacc atggcatccc ctatactagg
     1321 ttattggaaa attaagggcc ttgtgcaacc cactcgactt cttttggaat atcttgaaga
     1381 aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt
     1441 tgaattgggt ttggagtttc ccaatcttcc ttattatatt gatggtgatg ttaaattaac
     1501 acagtctatg gccatcatac gttatatagc tgacaagcac aacatgttgg gtggttgtcc
     1561 aaaagagcgt gcagagattt caatgcttga aggagcggtt ttggatatta gatacggtgt
     1621 ttcgagaatt gcatatagta aagactttga aactctcaaa gttgattttc ttagcaagct
     1681 acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt taaatggtga
     1741 tcatgtaacc catcctgact tcatgttgta tgacgctctt gatgttgttt tatacatgga
     1801 cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat
     1861 cccacaaatt gataagtact tgaaatccag caagtatata gcatggcctt tgcagggctg
     1921 gcaagccacg tttggtggtg gcgaccatcc tccaaaatcg gatgattacg atatcccaac
     1981 gaccgaaaac ctgtattttc agggtgtcga cggtaccatg gtgagcaagg gcgaggagct
     2041 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     2101 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     2161 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     2221 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     2281 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     2341 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     2401 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     2461 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     2521 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     2581 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccaagct
     2641 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     2701 cgggatcact ctcggcatgg acgagctgta caagggtacc atggcttccc catctggaaa
     2761 gggagctagg gctctggagg ctcctggatg cggaccacgg cccctggcca gagacctggt
     2821 ggatagcgtg gacgatgctg aggggctgta cgtggccgtg gaaagatgcc ccctgtgtaa
     2881 taccacacgg agaaggctga catgcgccaa gtgtgtgcag tccggggatt tcgtgtattt
     2941 tgacggacgc gatcgggaga gattcattga caagaaagaa aggctgtccc gcctgaagtc
     3001 taaacaggag gaatttcaga aagaggtgct gaaggccatg gagggcaagt ggatcactga
     3061 tcagctgagg tggaaaatca tgtcctgcaa gatgcgcatt gagcagctga aacagaccat
     3121 ctgtaaggga aacgaggaaa tggagaaaaa tagcgaaggc ctgctgaaga caaaagaaaa
     3181 gaatcagaag ctgtactccc gggctcagag acaccaggag aagaaagaaa aaattcagag
     3241 gcataaccgc aagctgggcg atctggtgga gaagaaaacc atcgacctga ggtctcacta
     3301 tgaacgcctg gccaatctgc gccggagcca tatcctggag ctgacatccg tgatcttccc
     3361 aattgaggaa gtgaaaactg gagtgaggga ccccgctgat gtgagctccg agagtgactc
     3421 agccatgacc tctagtacag tgagtaagct ggctgaagcc agaaggacta cctacctgtc
     3481 aggccgctgg gtgtgcgacg atcacaacgg cgatacaagc atctccatta ctgggccctg
     3541 gattagcctg cctaacaatg gcgactatag tgcctactat tcatgggtgg aggaaaagaa
     3601 aacaactcag gggccagata tggagcagtc taatcccgct tacactatca gtgccgctct
     3661 gtgctatgcc acccagctgg tgaacatcct gtctcatatt ctggacgtga acctgcccaa
     3721 gaaactgtgc aattcagagt tctgtggcga aaacctgagc aaacagaagt ttactagggc
     3781 tgtgaagaaa ctgaacgcca atattctgta cctgtgtttt agccagcacg tgaatctgga
     3841 tcagctgcag cctctgcata ccctgcgcaa cctgatgtat ctggtgtccc cttcaagcga
     3901 gcacctggga aggtctggac cattcgaagt gagagctgat ctggaggaaa gcatggagtt
     3961 tgtggaccct ggagtggccg gcgagtctga cgaaagtggg gatgagcggg tgtctgacga
     4021 ggaaactgat ctgggaaccg actgggaaaa cctgcctagt ccaagattct gtgacatccc
     4081 atctcagagt gtggaggtgt cacagagcca gtccacacag gcttcccccc ctattgcttc
     4141 ctctagtgct ggaggaatga tctcaagcgc cgctgcctca gtgaccagct ggtttaaggc
     4201 ctacacaggc cataggtgac tcgagactag tatcgcgata attcactcct caggtgcagg
     4261 ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag
     4321 atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt
     4381 ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct
     4441 cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt
     4501 agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag
     4561 gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga
     4621 cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa
     4681 aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca
     4741 tagctgtccc tcttctctta tggagatccc tcgacctgca gcccaagctt ggcgtaatca
     4801 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4861 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4921 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatcgatccg
     4981 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5041 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5101 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5161 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5221 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5281 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5341 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5401 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5461 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5521 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5581 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5641 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5701 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5761 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5821 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5881 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5941 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6001 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6061 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6121 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6181 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6241 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6301 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6361 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6421 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6481 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6541 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6601 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6661 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6721 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6781 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6841 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6901 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6961 ccacctgggt cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     7021 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     7081 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     7141 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     7201 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     7261 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     7321 catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac
     7381 tctccccatc tcccccccct ccccaccccc aattttgtat ttattt
//
