LOCUS       sequence-393058-        5886 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for the expression of 4X human ubiquitin in tandem (SMC interal
            no. 538).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5886)
  AUTHORS   Wurzer B, Zaffagnini G, Fracchiolla D, Turco E, Abert C, Romanov J, 
            Martens S
  TITLE     Oligomerization of p62 allows for selection of ubiquitinated cargo 
            and isolation membrane during selective autophagy.
  JOURNAL   Elife. 2015 Sep 28;4:e08941. doi: 10.7554/eLife.08941.
  PUBMED    26413874
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: "Elife."; 
            date: "2015-09-28"; volume: "4:e08941. doi"; pages: " 
            10.7554/eLife.08941"
COMMENT     Sequence Label: pGEX-4T1_4xUb
FEATURES             Location/Qualifiers
     source          1..5886
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(82..87,88..103,104..110)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    118..134
                     /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             157..810
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     768..790
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             817..834
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             847..1074
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             1075..1302
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             1303..1530
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             1531..1758
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     primer_bind     complement(1835..1857)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2002..2020)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2088..2192
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2193..2261,2262..3053)
                     /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(2411..2430)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3224..3812
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3713..3732
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3966..3983
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4056..4133
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4134..5216
                     /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(4153..4172)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5265..5270,5271..5288,5289..5295)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5303..5319
                     /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     5327..5343
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5327..5343
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5339..5866
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5359..5376)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5359..5375)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5368..5390)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg
       61 caaatattct gaaatgagct gttgacaatt aatcatcggc tcgtataatg tgtggaattg
      121 tgagcggata acaatttcac acaggaaaca gtattcatgt cccctatact aggttattgg
      181 aaaattaagg gccttgtgca acccactcga cttcttttgg aatatcttga agaaaaatat
      241 gaagagcatt tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg
      301 ggtttggagt ttcccaatct tccttattat attgatggtg atgttaaatt aacacagtct
      361 atggccatca tacgttatat agctgacaag cacaacatgt tgggtggttg tccaaaagag
      421 cgtgcagaga tttcaatgct tgaaggagcg gttttggata ttagatacgg tgtttcgaga
      481 attgcatata gtaaagactt tgaaactctc aaagttgatt ttcttagcaa gctacctgaa
      541 atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat atttaaatgg tgatcatgta
      601 acccatcctg acttcatgtt gtatgacgct cttgatgttg ttttatacat ggacccaatg
      661 tgcctggatg cgttcccaaa attagtttgt tttaaaaaac gtattgaagc tatcccacaa
      721 attgataagt acttgaaatc cagcaagtat atagcatggc ctttgcaggg ctggcaagcc
      781 acgtttggtg gtggcgacca tcctccaaaa tcggatctgg ttccgcgtgg atccccagga
      841 attcatatgc agatcttcgt gaagaccctg accggcaaga ccatcaccct agaggtggag
      901 cccagtgaca ccatcgagaa cgtgaaggcc aagatccagg ataaagaggg catcccccct
      961 gaccagcaga ggctgatctt tgccggcaag cagctggaag atggccgcac cctctctgat
     1021 tacaacatcc agaaggagtc aaccctgcac ctggtccttc gcctgagagg tggcatgcag
     1081 atcttcgtga agaccctgac cggcaagacc atcaccctgg aggtggagcc cagtgacacc
     1141 atcgagaacg tgaaggccaa gatccaggat aaagagggca tcccccctga ccagcagagg
     1201 ctgatctttg ccggcaagca gctggaagat ggccgcaccc tctctgatta caacatccag
     1261 aaagagtcaa ccctgcacct ggtcctccgt ctgaggggtg gcatgcagat cttcgtgaag
     1321 accctgactg gcaagaccat caccctggag gtggagccca gtgacaccat cgagaacgtg
     1381 aaggccaaga tccaggataa agagggcatc ccccctgacc agcagaggct gatctttgcc
     1441 ggcaagcagc tggaagatgg ccgcaccctc tctgattaca acatccagaa ggagtcaacc
     1501 ctgcacctgg tccttcgcct gagaggtggc atgcagatct tcgtgaagac cctgaccggc
     1561 aagaccatca ccttggaggt ggagcccagt gacaccatcg agaatgtgaa ggccaagatc
     1621 caggataaag agggcatccc ccctgaccag cagaggctga tctttgccgg caagcagcta
     1681 gaagatggcc gcactctctc tgattacaac atccagaaag agtcgaccct gcacctggtc
     1741 ctccgtctga ggggtggcta ttaactcgac tcgagcggcc gcatcgtgac tgactgacga
     1801 tctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     1861 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     1921 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     1981 ataattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     2041 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2101 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2161 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     2221 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2281 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2341 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2401 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     2461 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2521 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2581 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2641 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2701 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     2761 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2821 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2881 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2941 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3001 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3061 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3121 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3181 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3241 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3301 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3361 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3421 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3481 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3541 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3601 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3661 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3721 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3781 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3841 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3901 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3961 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4021 cgcatctgtg cggtatttca caccgcataa attccgacac catcgaatgg cgcaaaacct
     4081 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
     4141 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
     4201 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     4261 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
     4321 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
     4381 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     4441 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     4501 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     4561 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     4621 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     4681 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     4741 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
     4801 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
     4861 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
     4921 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
     4981 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     5041 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     5101 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
     5161 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     5221 aacgcaatta atgtaagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     5281 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     5341 gaccatgatt acggattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     5401 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     5461 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgctt
     5521 tgcctggttt ccggcaccag aagcggtgcc ggaaagctgg ctggagtgcg atcttcctga
     5581 ggccgatact gtcgtcgtcc cctcaaactg gcagatgcac ggttacgatg cgcccatcta
     5641 caccaacgtg acctatccca ttacggtcaa tccgccgttt gttcccacgg agaatccgac
     5701 gggttgttac tcgctcacat ttaatgttga tgaaagctgg ctacaggaag gccagacgcg
     5761 aattattttt gatggcgttg gaattagctt atcgactgca cggtgcacca atgcttctgg
     5821 cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt
     5881 cgtgtc
//
