LOCUS       sequence-373589-        6134 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for the expression and purification of of ATG4B. Internal 
            reference: SMC1392.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6134)
  AUTHORS   Fracchiolla D, Chang C, Hurley JH, Martens S
  TITLE     A PI3K-WIPI2 positive feedback loop allosterically activates LC3 
            lipidation in autophagy.
  JOURNAL   J Cell Biol. 2020 Jul 6;219(7). pii: 151802. doi: 
            10.1083/jcb.201912098.
  PUBMED    32437499
REFERENCE   2  (bases 1 to 6134)
  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: "J Cell 
            Biol."; date: "2020-07-6"; volume: "219(7). pii: 151802. doi"; 
            pages: " 10.1083/jcb.201912098"
COMMENT     Sequence Label: pGEX-4T1_ATG4B_BamHI_NotI
FEATURES             Location/Qualifiers
     source          1..6134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(2184..2206)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2351..2369)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2437..2541
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2542..2610,2611..3402)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2760..2779)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3573..4161
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4062..4081
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4315..4332
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4405..4482
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4483..5565
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4502..4521)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5614..5619,5620..5637,5638..5644)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5652..5668
                     /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)."
     primer_bind     5676..5692
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5676..5692
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5688..6134,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5708..5725)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5708..5724)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5717..5739)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatccggagg aatggacgca gctactctga
      961 cctacgacac tctccggttt gctgagtttg aagattttcc tgagacctca gagcccgttt
     1021 ggatactggg tagaaaatac agcattttca cagaaaagga cgagatcttg tctgatgtgg
     1081 catctagact ttggtttaca tacaggaaaa actttccagc cattgggggg acaggcccca
     1141 cctcggacac aggctggggc tgcatgctgc ggtgtggaca gatgatcttt gcccaagccc
     1201 tggtgtgccg gcacctaggc cgagattgga ggtggacaca aaggaagagg cagccagaca
     1261 gctacttcag cgtcctcaac gcattcatcg acaggaagga cagttactac tccattcacc
     1321 agatagcgca aatgggagtt ggcgaaggca agtccatagg ccagtggtac gggcccaaca
     1381 ctgtcgccca ggtcctgaag aagcttgctg tcttcgatac gtggagctcc ttggcggtcc
     1441 acattgcaat ggacaacact gttgtgatgg aggaaatcag aaggttgtgc aggaccagcg
     1501 ttccctgtgc aggcgccact gcgtttcctg cagattccga ccggcactgc aacggattcc
     1561 ctgccggagc tgaggtcacc aacaggccgt cgccatggag acccctggta cttctcattc
     1621 ccctgcgcct ggggctcacg gacatcaacg aggcctacgt ggagacgctg aagcactgct
     1681 tcatgatgcc ccagtccctg ggcgtcatcg gagggaagcc caacagcgcc cactacttca
     1741 tcggctacgt tggtgaggag ctcatctacc tggaccccca caccacgcag ccagccgtgg
     1801 agcccactga tggctgcttc atcccggacg agagcttcca ctgccagcac ccgccgtgcc
     1861 gcatgagcat cgcggagctt gacccgtcca tcgctgtggg gtttttctgt aagactgaag
     1921 atgacttcaa tgattggtgc cagcaagtca aaaagctgtc tctgcttgga ggtgccctgc
     1981 ccatgtttga gctggtggag cagcagcctt cacatctggc ctgccccgac gtcctgaacc
     2041 tgtccctaga ttcttctgat gtagagcgac tggaaagatt cttcgactca gaagatgaag
     2101 actttgaaat cctgtccctt tgagcggccg catcgtgact gactgacgat ctgcctcgcg
     2161 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     2221 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     2281 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta taattcttga
     2341 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     2401 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2461 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2521 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     2581 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     2641 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     2701 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     2761 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata
     2821 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     2881 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     2941 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3001 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     3061 gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact
     3121 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3181 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3241 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     3301 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3361 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     3421 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     3481 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3541 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3601 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3661 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     3721 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3781 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3841 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3901 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3961 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4021 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4081 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4141 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4201 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4261 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4321 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     4381 ggtatttcac accgcataaa ttccgacacc atcgaatggc gcaaaacctt tcgcggtatg
     4441 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta
     4501 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
     4561 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
     4621 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
     4681 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
     4741 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
     4801 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
     4861 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
     4921 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
     4981 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     5041 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     5101 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     5161 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
     5221 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     5281 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
     5341 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     5401 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     5461 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     5521 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     5581 tgtaagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     5641 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     5701 cggattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     5761 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     5821 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcttt gcctggtttc
     5881 cggcaccaga agcggtgccg gaaagctggc tggagtgcga tcttcctgag gccgatactg
     5941 tcgtcgtccc ctcaaactgg cagatgcacg gttacgatgc gcccatctac accaacgtga
     6001 cctatcccat tacggtcaat ccgccgtttg ttcccacgga gaatccgacg ggttgttact
     6061 cgctcacatt taatgttgat gaaagctggc tacaggaagg ccagacgcga attatttttg
     6121 atggcgttgg aatt
//
