LOCUS       sequence-443802-        5250 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression construct for GST-Tagged Ubiquitin production 
            and purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5250)
  TITLE     STAR-protocols--Evaluating the E3 Ligase Activity of BRCA1-BARD1 and
            its Variants by Nucleosomal Histone Ubiquitylation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5250)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pGEX-6p1-GST-Ubiquitin
FEATURES             Location/Qualifiers
     source          1..5250
                     /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="MSPILGYWKITGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPL*GWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             939..971
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             981..1208
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     primer_bind     complement(1301..1323)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1468..1486)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1554..1658
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1659..1727,1728..2519)
                     /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(1877..1896)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2690..3277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3179..3198
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3431..3448
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3521..3598
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3599..4681
                     /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(3618..3637)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4730..4735,4736..4753,4754..4760)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4768..4784
                     /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     4792..4808
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4792..4808
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4824..4841)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4824..4840)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4833..4855)
                     /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 gaaaattacg 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 cctttgtagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggat ggctagcatg actggtggac
      961 agcaaatggg tcgcggatcc atgcagatct tcgtcaagac gttaaccggt aaaaccataa
     1021 ctctggaagt tgaaccatcc gataccatcg aaaacgttaa ggctaaaatt caagacaagg
     1081 aaggaattcc acctgatcaa caaagattga tctttgccgg gaagcagctc gaagacggta
     1141 gaacgctgtc tgattacaac attcagaagg agtcgacctt acatcttgtc ttaagactaa
     1201 gaggtggttg aggatccccg gaattcccgg gtcgactcga gcggccgcat cgtgactgac
     1261 tgacgatctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     1321 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     1381 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
     1441 gagtgtataa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     1501 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     1561 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1621 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     1681 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     1741 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     1801 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1861 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc
     1921 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1981 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     2041 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     2101 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     2161 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     2221 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     2281 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     2341 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     2401 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     2461 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     2521 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     2581 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     2641 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2701 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2761 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2821 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2881 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2941 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3001 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3061 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3121 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3181 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3241 ttttgtgatg ctcgtcaggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3301 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3361 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3421 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     3481 ccttacgcat ctgtgcggta tttcacaccg cataaattcc gacaccatcg aatggcgcaa
     3541 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
     3601 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     3661 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
     3721 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     3781 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     3841 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     3901 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     3961 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     4021 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     4081 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     4141 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     4201 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
     4261 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
     4321 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
     4381 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
     4441 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     4501 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagt tgttgcccgt
     4561 ctcactggtg aaaagaaaaa ccacgctagc gcccaatacg caaaccgcct ctccccgcgc
     4621 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     4681 agcgcaacgc aattaatgta agttagctca ctcattaggc accccaggct ttacacttta
     4741 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     4801 gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     4861 cctggcgtta cccaacttaa tcgccttgca gcacatctcc ctttcgccag ctggcgtaat
     4921 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     4981 cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt
     5041 cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta cgatgcgccc
     5101 atctacacca acgtgaccta tcccattacg gtcaatccgc cgtttgttcc cacggagaat
     5161 ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca ggaaggccag
     5221 acgcgaatta tttttgatgg cgttggaatt
//
