LOCUS       sequence-337564-        6943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the purifcation of GFP-ULK1 complex .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6943)
  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 6943)
  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-TEVcs-GFP-ATG101
FEATURES             Location/Qualifiers
     source          1..6943
                     /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             1760..2416
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MASPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2374..2396
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2444..2464
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2471..2473,2474..2476,2477..3187)
                     /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(2516..2537)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2777..2796)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3124..3145
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3965..3984)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4030..4085
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4084..4103)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4446..4462)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4446..4462)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4459..4481)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4470..4486
                     /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(4494..4500,4501..4518,4519..4524))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4539..4560
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4619..4814
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4665..4800
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4820..4954
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4857..4876)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4911..4930
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5022..5039)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5193..5781)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5273..5292)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5952..6743,6744..6812))
                     /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     6575..6594
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6813..6917)
                     /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 gcgccrggsg gggsggggsg gggsgrgggg
      541 sggggsgggg sgaggcggag aggtgcggcg 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 tggcatcccc tatactaggt tattggaaaa ttaagggcct
     1801 tgtgcaaccc actcgacttc ttttggaata tcttgaagaa aaatatgaag agcatttgta
     1861 tgagcgcgat gaaggtgata aatggcgaaa caaaaagttt gaattgggtt tggagtttcc
     1921 caatcttcct tattatattg atggtgatgt taaattaaca cagtctatgg ccatcatacg
     1981 ttatatagct gacaagcaca acatgttggg tggttgtcca aaagagcgtg cagagatttc
     2041 aatgcttgaa ggagcggttt tggatattag atacggtgtt tcgagaattg catatagtaa
     2101 agactttgaa actctcaaag ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt
     2161 cgaagatcgt ttatgtcata aaacatattt aaatggtgat catgtaaccc atcctgactt
     2221 catgttgtat gacgctcttg atgttgtttt atacatggac ccaatgtgcc tggatgcgtt
     2281 cccaaaatta gtttgtttta aaaaacgtat tgaagctatc ccacaaattg ataagtactt
     2341 gaaatccagc aagtatatag catggccttt gcagggctgg caagccacgt ttggtggtgg
     2401 cgaccatcct ccaaaatcgg atgattacga tatcccaacg accgaaaacc tgtattttca
     2461 gggtggtacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     2521 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     2581 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     2641 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     2701 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     2761 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     2821 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     2881 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     2941 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     3001 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3061 cgacaaccac tacctgagca cccagtccaa gctgagcaaa gaccccaacg agaagcgcga
     3121 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     3181 gtacaagggt accatgaatt gccggtcaga ggtcctggaa gtgagcgtgg aaggtagaca
     3241 ggtggaagag gctatgctgg ccgtgctgca tacagtgctg ctgcacagat caactggcaa
     3301 gttccattac aagaaagaag gaacatattc catcggaacc gtgggtactc aggacgtcga
     3361 ttgcgacttc attgatttta cttacgtgcg cgtcagctcc gaggaactgg acagagcact
     3421 ccgtaaggtg gtcggagaat tcaaagatgc cctgaggaac agtggcggag acggtctggg
     3481 ccagatgagc ctcgagttct atcagaagaa aaagtctaga tggccatttt cagatgaatg
     3541 tatcccctgg gaagtgtgga ccgtgaaggt ccacgtggtc gccctggcta ctgagcagga
     3601 acggcagatc tgccgcgaga aagtgggcga aaagctctgt gagaagatca tcaacatcgt
     3661 ggaagtcatg aaccgtcatg agtacctgcc caagatgcct acacagagcg aggtggataa
     3721 tgtcttcgac accggtctgc gggatgtgca gccctacctc tataagatct cctttcagat
     3781 tacagacgct ctgggcacct ctgtcaccac tacaatgcgc aggctcatca aagacaccct
     3841 ggcactctaa cccgggtgat atcttaatta attaactcga ggaattcact cctcaggtgc
     3901 aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact
     3961 gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga
     4021 cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc
     4081 tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg
     4141 tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa
     4201 gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct
     4261 tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc
     4321 taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag
     4381 tcatagctgt ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa
     4441 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4501 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4561 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc
     4621 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     4681 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4741 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4801 taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa
     4861 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4921 caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc
     4981 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5041 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5101 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5161 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5221 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5281 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5341 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5401 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5461 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5521 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5581 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     5641 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5701 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5761 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5821 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5881 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5941 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6001 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6061 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     6121 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6181 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6241 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6301 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6361 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6421 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6481 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6541 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6601 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6661 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6721 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6781 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6841 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6901 caaatagggg ttccgcgcac atttccccga aaagtgccac ctg
//
