LOCUS       sequence-326704-        6035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of a GST fusion of Human E6AP Hect domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6035)
  AUTHORS   Ronchi VP, Haas AL
  TITLE     Measuring rates of ubiquitin chain formation as a functional readout
            of ligase activity.
  JOURNAL   Methods Mol Biol. 2012;832:197-218. doi: 
            10.1007/978-1-61779-474-2_14.
  PUBMED    22350887
REFERENCE   2  (bases 1 to 6035)
  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: "Methods Mol
            Biol."; date: "2012"; volume: "832:197-218. doi"; pages: " 
            10.1007/978-1-61779-474-2_14"
COMMENT     Sequence Label: pGEX-E6AP Hect
FEATURES             Location/Qualifiers
     source          1..6035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     356..375
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     609..626
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        699..776
                     /gene="lacI"
                     /label=lacI promoter
     CDS             777..1859
                     /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(796..815)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1908..1913,1914..1931,1932..1938)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1946..1962
                     /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     1970..1986
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1970..1986
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1982..2509
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(2002..2019)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2002..2018)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2011..2033)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(2611..2616,2617..2632,2633..2639)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2647..2663
                     /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             2686..3339
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     3297..3319
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             3346..3363
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(4513..4535)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4680..4698)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4766..4870
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4871..4939,4940..5731)
                     /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(5089..5108)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5902..6035,1..455)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
       61 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
      121 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
      181 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
      241 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
      301 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
      361 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
      421 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
      481 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
      541 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
      601 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
      661 ttacgcatct gtgcggtatt tcacaccgca taaattccga caccatcgaa tggcgcaaaa
      721 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
      781 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
      841 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
      901 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
      961 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     1021 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     1081 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     1141 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     1201 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     1261 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     1321 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     1381 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     1441 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     1501 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     1561 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     1621 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     1681 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     1741 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     1801 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     1861 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cccaggcttt acactttatg
     1921 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     1981 tatgaccatg attacggatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     2041 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     2101 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     2161 ctttgcctgg tttccggcac cagaagcggt gccggaaagc tggctggagt gcgatcttcc
     2221 tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat
     2281 ctacaccaac gtgacctatc ccattacggt caatccgccg tttgttccca cggagaatcc
     2341 gacgggttgt tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac
     2401 gcgaattatt tttgatggcg ttggaattag cttatcgact gcacggtgca ccaatgcttc
     2461 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata
     2521 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa
     2581 cggttctggc aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt
     2641 gtggaattgt gagcggataa caatttcaca caggaaacag tattcatgtc ccctatacta
     2701 ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga atatcttgaa
     2761 gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg aaacaaaaag
     2821 tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga tgttaaatta
     2881 acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt gggtggttgt
     2941 ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat tagatacggt
     3001 gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt tcttagcaag
     3061 ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata tttaaatggt
     3121 gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt tttatacatg
     3181 gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg tattgaagct
     3241 atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc tttgcagggc
     3301 tggcaagcca cgtttggtgg tggcgaccat cctccaaaat cggatctggt tccgcgtgga
     3361 tccccgaatt cgatgcagtt gaatccatat ttgagactca aagttagacg tgaccatatc
     3421 atagatgatg cacttgtccg gctagagatg atcgctatgg aaaatcctgc agacttgaag
     3481 aagcagttgt atgtggaatt tgaaggagaa caaggagttg atgagggagg tgtttccaaa
     3541 gaattttttc agctggttgt ggaggaaatc ttcaatccag atattggtat gttcacatac
     3601 gatgaatcta caaaattgtt ttggtttaat ccatcttctt ttgaaactga gggtcagttt
     3661 actctgattg gcatagtact gggtctggct atttacaata actgtatact ggatgtacat
     3721 tttcccatgg ttgtctacag gaagctaatg gggaaaaaag gaacttttcg tgacttggga
     3781 gactctcacc cagttctata tcagagttta aaagatttat tggagtatga agggaatgtg
     3841 gaagatgaca tgatgatcac tttccagata tcacagacag atctttttgg taacccaatg
     3901 atgtatgatc taaaggaaaa tggtgataaa attccaatta caaatgaaaa caggaaggaa
     3961 tttgtcaatc tttattctga ctacattctc aataaatcag tagaaaaaca gttcaaggct
     4021 tttcggagag gttttcatat ggtgaccaat gaatctccct taaagtactt attcagacca
     4081 gaagaaattg aattgcttat atgtggaagc cggaatctag atttccaagc actagaagaa
     4141 actacagaat atgacggtgg ctataccagg gactctgttc tgattaggga gttctgggaa
     4201 atcgttcatt catttacaga tgaacagaaa agactcttct tgcagtttac aacgggcaca
     4261 gacagagcac ctgtgggagg actaggaaaa ttaaagatga ttatagccaa aaatggccca
     4321 gacacagaaa ggttacctac atctcatact tgctttaatg tgcttttact tccggaatac
     4381 tcaagcaaag aaaaacttaa agagagattg ttgaaggcca tcacgtatgc caaaggattt
     4441 ggcatgctgt aagcggccgc atcgtgactg actgacgatc tgcctcgcgc gtttcggtga
     4501 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     4561 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     4621 cgcagccatg acccagtcac gtagcgatag cggagtgtat aattcttgaa gacgaaaggg
     4681 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     4741 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4801 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4861 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4921 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4981 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5041 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5101 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5161 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5221 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5281 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5341 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5401 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     5461 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     5521 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     5581 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     5641 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5701 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     5761 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5821 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5881 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5941 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6001 ttttccgaag gtaactggct tcagcagagc gcaga
//
