LOCUS       sequence-337506-        6667 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the purifcation of phosphorylation mimic  protein .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6667)
  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 6667)
  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-OPTNS177DS473D
FEATURES             Location/Qualifiers
     source          1..6667
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(530..535,536..553,554..560)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    568..584
                     /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     592..608
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     592..608
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             604..1131
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(624..641)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(624..640)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(633..655)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(1233..1238,1239..1254,1255..1261)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1269..1285
                     /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             1308..1961
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1919..1941
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2285..2296
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3767..3789)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3934..3952)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4020..4124
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4125..4193,4194..4985)
                     /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(4343..4362)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5156..5744
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5645..5664
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5898..5915
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5988..6065
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(6066..6667,1..481)
                     /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(6085..6104)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
       61 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
      121 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
      181 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
      241 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
      301 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
      361 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
      421 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
      481 agcgcaacgc aattaatgta agttagctca ctcattaggc accccaggct ttacacttta
      541 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
      601 gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
      661 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
      721 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
      781 cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt
      841 cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta cgatgcgccc
      901 atctacacca acgtgaccta tcccattacg gtcaatccgc cgtttgttcc cacggagaat
      961 ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca ggaaggccag
     1021 acgcgaatta tttttgatgg cgttggaatt agcttatcga ctgcacggtg caccaatgct
     1081 tctggcgtca ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca
     1141 taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat
     1201 aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatcgg ctcgtataat
     1261 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agtattcatg tcccctatac
     1321 taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg gaatatcttg
     1381 aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg cgaaacaaaa
     1441 agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt gatgttaaat
     1501 taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg ttgggtggtt
     1561 gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat attagatacg
     1621 gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat tttcttagca
     1681 agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca tatttaaatg
     1741 gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt gttttataca
     1801 tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa cgtattgaag
     1861 ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg cctttgcagg
     1921 gctggcaagc cacgtttggt ggtggcgacc atcctccaaa aatcgatatg tcccatcaac
     1981 ctctcagctg cctcactgaa aaggaggaca gccccagtga aagcacagga aatggacccc
     2041 cccacctggc ccacccaaac ctggacacgt ttaccccgga ggagctgctg cagcagatga
     2101 aagagctcct gaccgagaac caccagctga aagaagccat gaagctaaat aatcaagcca
     2161 tgaaagggag atttgaggag ctttcggcct ggacagagaa acagaaggaa gaacgccagt
     2221 tttttgagat acagagcaaa gaagcaaaag agcgtctaat ggccttgagt catgagaatg
     2281 agaaattgaa ggaagagctt ggaaaactaa aagggaaatc agaaaggtca tctgaggacc
     2341 ccactgatga ctccaggctt cccagggccg aagcggagca ggaaaaggac cagctcagga
     2401 cccaggtggt gaggctacaa gcagagaagg cagacctgtt gggcatcgtg tctgaactgc
     2461 agctcaagct gaactccagc ggctcctcag aagatgattt tgttgaaatt aggatggctg
     2521 aaggagaagc agaagggtca gtaaaagaaa tcaagcatag tcctgggccc acgagaacag
     2581 tctccactgg cacggcattg tctaaatata ggagcagatc tgcagatggg gccaagaatt
     2641 acttcgaaca tgaggagtta actgtgagcc agctcctgct gtgcctaagg gaagggaatc
     2701 agaaggtgga gagacttgaa gttgcactca aggaggccaa agaaagagtt tcagattttg
     2761 aaaagaaaac aagtaatcgt tctgagattg aaacccagac agaggggagc acagagaaag
     2821 agaatgatga agagaaaggc ccggagactg ttggaagcga agtggaagca ctgaacctcc
     2881 aggtgacatc tctgtttaag gagcttcaag aggctcatac aaaactcagc gaagctgagc
     2941 taatgaagaa gagacttcaa gaaaagtgtc aggcccttga aaggaaaaat tctgcaattc
     3001 catcagagtt gaatgaaaag caagagcttg tttatactaa caaaaagtta gagctacaag
     3061 tggaaagcat gctatcagaa atcaaaatgg aacaggctaa aacagaggat gaaaagtcca
     3121 aattaactgt gctacagatg acacacaaca agcttcttca agaacataat aatgcattga
     3181 aaacaattga ggaactaaca agaaaagagt cagaaaaagt ggacagggca gtgctgaagg
     3241 aactgagtga aaaactggaa ctggcagaga aggctctggc ttccaaacag ctgcaaatgg
     3301 atgaaatgaa gcaaaccatt gccaagcagg aagaggacct ggaaaccatg accatcctca
     3361 gggctcagat ggaagtttac tgtgatgatt ttcatgctga aagagcagcg agagagaaaa
     3421 ttcatgagga aaaggagcaa ctggcattgc agctggcagt tctgctgaaa gagaatgatg
     3481 ctttcgaaga cggaggcagg cagtccttga tggagatgca gagtcgtcat ggggcgagaa
     3541 caagtgactc tgaccagcag gcttaccttg ttcaaagagg agctgaggac agggactggc
     3601 ggcaacagcg gaatattccg attcattcct gccccaagtg tggagaggtt ctgcctgaca
     3661 tagacacgtt acagattcac gtgatggatt gcatcattta actcgagcgg ccgcatcgtg
     3721 actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     3781 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     3841 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     3901 atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     3961 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     4021 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4081 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4141 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4201 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4261 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4321 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     4381 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     4441 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4501 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     4561 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4621 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     4681 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     4741 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     4801 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     4861 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     4921 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4981 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5041 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5101 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5161 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5221 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5281 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     5341 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5401 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5461 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5521 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5581 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5641 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5701 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5761 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5821 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5881 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     5941 attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac accatcgaat
     6001 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     6061 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     6121 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     6181 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     6241 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     6301 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     6361 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     6421 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     6481 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     6541 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     6601 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     6661 ctggctg
//
