LOCUS       sequence-382515-        5272 bp    DNA     circular SYN 15-APR-2025
DEFINITION  In vitro ubiquitination assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5272)
  AUTHORS   Smith CE, Tsai YC, Liang YH, Khago D, Mariano J, Li J, Tarasov SG, 
            Gergel E, Tsai B, Villaneuva M, Clapp ME, Magidson V, Chari R, Byrd 
            RA, Ji X, Weissman AM
  TITLE     A structurally conserved site in AUP1 binds the E2 enzyme UBE2G2 and
            is essential for ER-associated degradation.
  JOURNAL   PLoS Biol. 2021 Dec 8;19(12):e3001474. doi: 
            10.1371/journal.pbio.3001474. eCollection 2021 Dec.
  PUBMED    34879065
REFERENCE   2  (bases 1 to 5272)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS 
            Biol."; date: "2021-12-8"; volume: "19(12):e3001474. doi"; pages: " 
            10.1371/journal.pbio.3001474. eCollection 2021 Dec"
COMMENT     Sequence Label: pGEX-6P-1-Cys-HA-Ubiquitin
FEATURES             Location/Qualifiers
     source          1..5272
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(177..182,183..198,199..205)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    213..229
                     /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             252..905
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     863..885
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             912..935
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             948..974
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             975..1001
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             975..1001
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1008..1232
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="QIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFA
                     GKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     primer_bind     complement(1316..1338)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1483..1501)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1569..1673
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1674..1742,1743..2534)
                     /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(1892..1911)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2705..3293
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3194..3213
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3447..3464
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3537..3614
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3615..4697
                     /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(3634..3653)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4746..4751,4752..4769,4770..4776)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4784..4800
                     /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     4808..4824
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4808..4824
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4820..5272,1..75)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4840..4857)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4840..4856)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4849..4871)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc tgtggtatgg
       61 ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca aggcgcactc ccgttctgga
      121 taatgttttt tgcgccgaca tcataacggt tctggcaaat attctgaaat gagctgttga
      181 caattaatca tcggctcgta taatgtgtgg aattgtgagc ggataacaat ttcacacagg
      241 aaacagtatt catgtcccct atactaggtt attggaaaat taagggcctt gtgcaaccca
      301 ctcgacttct tttggaatat cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg
      361 aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttcctt
      421 attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt tatatagctg
      481 acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca atgcttgaag
      541 gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa gactttgaaa
      601 ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt
      661 tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatg
      721 acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc ccaaaattag
      781 tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg aaatccagca
      841 agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc gaccatcctc
      901 caaaatcgga tctggaagtt ctgttccagg ggcccctggg atcctgctac ccatacgatg
      961 ttcctgatta cgcttacccc tacgacgtgc ccgactacgc cggatcccag attttcgtca
     1021 agactttgac cggtaaaacc ataacattgg aagttgaatc ttccgatacc atcgacaacg
     1081 ttaagtcgaa aattcaagac aaggaaggta tccctccaga tcaacaaaga ttgatctttg
     1141 ccggtaagca gctagaagac ggtagaacgc tgtctgatta caacattcag aaggagtcca
     1201 ccttacatct tgtgctaagg ctaagaggtg gttgagaatt cccgggtcga ctcgagcggc
     1261 cgcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     1321 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     1381 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     1441 cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg atacgcctat
     1501 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     1561 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1621 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     1681 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     1741 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     1801 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     1861 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     1921 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1981 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     2041 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     2101 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     2161 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     2221 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     2281 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     2341 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     2401 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     2461 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     2521 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     2581 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     2641 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     2701 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     2761 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     2821 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     2881 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     2941 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3001 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3061 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3121 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3181 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3241 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3301 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3361 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3421 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     3481 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata aattccgaca
     3541 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     3601 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     3661 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     3721 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     3781 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     3841 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     3901 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     3961 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     4021 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     4081 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     4141 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     4201 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     4261 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     4321 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     4381 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     4441 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     4501 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     4561 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac gctagcgccc aatacgcaaa
     4621 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     4681 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccc
     4741 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     4801 tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg ttttacaacg
     4861 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     4921 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     4981 cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc cggaaagctg
     5041 gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact ggcagatgca
     5101 cggttacgat gcgcccatct acaccaacgt gacctatccc attacggtca atccgccgtt
     5161 tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg atgaaagctg
     5221 gctacaggaa ggccagacgc gaattatttt tgatggcgtt ggaattagct ta
//
