LOCUS       sequence-337515-        5886 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the purifcation of tetra linear ubiquitin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5886)
  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 5886)
  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-Ub4
FEATURES             Location/Qualifiers
     source          1..5886
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(74..79,80..95,96..102)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    110..126
                     /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             149..802
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     760..782
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             809..826
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             839..1066
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             1067..1294
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             1295..1522
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             1523..1750
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     primer_bind     complement(1827..1849)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1994..2012)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2080..2184
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2185..2253,2254..3045)
                     /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(2403..2422)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3216..3804
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3705..3724
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3958..3975
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4048..4125
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4126..5208
                     /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(4145..4164)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5257..5262,5263..5280,5281..5287)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5295..5311
                     /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     5319..5335
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5319..5335
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5331..5858
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5351..5368)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5351..5367)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5360..5382)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cgcactcccg ttctggataa tgttttttgc gccgacatca taacggttct ggcaaatatt
       61 ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga
      121 taacaatttc acacaggaaa cagtattcat gtcccctata ctaggttatt ggaaaattaa
      181 gggccttgtg caacccactc gacttctttt ggaatatctt gaagaaaaat atgaagagca
      241 tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga
      301 gtttcccaat cttccttatt atattgatgg tgatgttaaa ttaacacagt ctatggccat
      361 catacgttat atagctgaca agcacaacat gttgggtggt tgtccaaaag agcgtgcaga
      421 gatttcaatg cttgaaggag cggttttgga tattagatac ggtgtttcga gaattgcata
      481 tagtaaagac tttgaaactc tcaaagttga ttttcttagc aagctacctg aaatgctgaa
      541 aatgttcgaa gatcgtttat gtcataaaac atatttaaat ggtgatcatg taacccatcc
      601 tgacttcatg ttgtatgacg ctcttgatgt tgttttatac atggacccaa tgtgcctgga
      661 tgcgttccca aaattagttt gttttaaaaa acgtattgaa gctatcccac aaattgataa
      721 gtacttgaaa tccagcaagt atatagcatg gcctttgcag ggctggcaag ccacgtttgg
      781 tggtggcgac catcctccaa aatcggatct ggttccgcgt ggatccccag gaattcatat
      841 gcagatcttc gtgaagaccc tgaccggcaa gaccatcacc ctagaggtgg agcccagtga
      901 caccatcgag aacgtgaagg ccaagatcca ggataaagag ggcatccccc ctgaccagca
      961 gaggctgatc tttgccggca agcagctgga agatggccgc accctctctg attacaacat
     1021 ccagaaggag tcaaccctgc acctggtcct tcgcctgaga ggtggcatgc agatcttcgt
     1081 gaagaccctg accggcaaga ccatcaccct ggaggtggag cccagtgaca ccatcgagaa
     1141 cgtgaaggcc aagatccagg ataaagaggg catcccccct gaccagcaga ggctgatctt
     1201 tgccggcaag cagctggaag atggccgcac cctctctgat tacaacatcc agaaagagtc
     1261 aaccctgcac ctggtcctcc gtctgagggg tggcatgcag atcttcgtga agaccctgac
     1321 tggcaagacc atcaccctgg aggtggagcc cagtgacacc atcgagaacg tgaaggccaa
     1381 gatccaggat aaagagggca tcccccctga ccagcagagg ctgatctttg ccggcaagca
     1441 gctggaagat ggccgcaccc tctctgatta caacatccag aaggagtcaa ccctgcacct
     1501 ggtccttcgc ctgagaggtg gcatgcagat cttcgtgaag accctgaccg gcaagaccat
     1561 caccttggag gtggagccca gtgacaccat cgagaatgtg aaggccaaga tccaggataa
     1621 agagggcatc ccccctgacc agcagaggct gatctttgcc ggcaagcagc tagaagatgg
     1681 ccgcactctc tctgattaca acatccagaa agagtcgacc ctgcacctgg tcctccgtct
     1741 gaggggtggc tattaactcg actcgagcgg ccgcatcgtg actgactgac gatctgcctc
     1801 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     1861 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     1921 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtataattct
     1981 tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     2041 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     2101 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     2161 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     2221 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     2281 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     2341 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     2401 ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc
     2461 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     2521 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     2581 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     2641 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     2701 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     2761 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     2821 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     2881 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     2941 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     3001 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     3061 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     3121 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     3181 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3241 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3301 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3361 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3421 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3481 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3541 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3601 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3661 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3721 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3781 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3841 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3901 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3961 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     4021 tgcggtattt cacaccgcat aaattccgac accatcgaat ggcgcaaaac ctttcgcggt
     4081 atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg
     4141 ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac
     4201 caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg
     4261 aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc
     4321 gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct
     4381 cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa
     4441 gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac
     4501 tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg
     4561 ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac
     4621 ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta
     4681 gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc
     4741 actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt
     4801 tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc
     4861 aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt
     4921 gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg
     4981 ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg
     5041 caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa
     5101 agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     5161 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     5221 taatgtaagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     5281 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     5341 ttacggattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     5401 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     5461 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc tttgcctggt
     5521 ttccggcacc agaagcggtg ccggaaagct ggctggagtg cgatcttcct gaggccgata
     5581 ctgtcgtcgt cccctcaaac tggcagatgc acggttacga tgcgcccatc tacaccaacg
     5641 tgacctatcc cattacggtc aatccgccgt ttgttcccac ggagaatccg acgggttgtt
     5701 actcgctcac atttaatgtt gatgaaagct ggctacagga aggccagacg cgaattattt
     5761 ttgatggcgt tggaattagc ttatcgactg cacggtgcac caatgcttct ggcgtcaggc
     5821 agccatcgga agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc
     5881 tcaagg
//
