LOCUS       sequence-494555-        5417 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-term GST tag E2 variant selected for RNF14.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5417)
  TITLE     E2 Variants for Probing E3 Ubiquitin Ligase Activities
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5417)
  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            "Unpublished"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: GST-tag RNF14 binding E2 variant L3R14-1
FEATURES             Location/Qualifiers
     source          1..5417
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..756
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     regulatory      758..767
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1285..1307)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1452..1470)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1538..1642
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1643..1711,1712..2503)
                     /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(1861..1880)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2674..3262
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3163..3182
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3416..3433
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3506..3583
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3584..4666
                     /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(3603..3622)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4715..4720,4721..4738,4739..4745)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4753..4769
                     /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     4777..4793
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4777..4793
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4789..5316
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4809..4826)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4809..4825)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4818..4840)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatgaaaa cctgtatttt cagggatcca tggcggccag caggaggctg
      781 atgaaggagc ttgaagaaat ccgcaaagca gggatgaaaa acttccgtaa catccaggtt
      841 gatgaagcta atttattgac ttggcaaggg cttattgttc ctgacaaccc tccatatgat
      901 aagggagcct tcagaatcga aatcaacttt ccagcagagt acccattcaa accaccgaag
      961 atcacattta aaacaaagat ctatcaccca aacatcgacg aaaaggggca ggtctgtctg
     1021 ccagtaatta gtgccgaaaa gtggcagcgg gagacctgga ccgaccaagt aatccagtcc
     1081 ctcatagcac tggtgaatga cccccagcct gagcacccgc ttcgggctga cctagctgaa
     1141 gaatactcta aggaccgtaa aaaattcgca aagaatgctg aagagtttac aaagaaatat
     1201 ggggaaaagc gacctgtgga ctaagcggcc gcatcgtgac tgactgacga tctgcctcgc
     1261 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     1321 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     1381 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt ataattcttg
     1441 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     1501 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1561 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     1621 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     1681 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     1741 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     1801 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     1861 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     1921 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     1981 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     2041 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     2101 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     2161 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     2221 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     2281 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     2341 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     2401 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     2461 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     2521 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     2581 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     2641 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2701 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2761 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2821 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2881 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2941 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3001 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3061 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3121 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3181 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3241 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3301 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3361 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3421 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     3481 cggtatttca caccgcataa attccgacac catcgaatgg cgcaaaacct ttcgcggtat
     3541 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     3601 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     3661 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     3721 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     3781 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     3841 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     3901 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     3961 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     4021 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     4081 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     4141 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     4201 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     4261 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     4321 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
     4381 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     4441 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     4501 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     4561 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     4621 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     4681 atgtaagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     4741 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     4801 acggattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     4861 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     4921 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgctt tgcctggttt
     4981 ccggcaccag aagcggtgcc ggaaagctgg ctggagtgcg atcttcctga ggccgatact
     5041 gtcgtcgtcc cctcaaactg gcagatgcac ggttacgatg cgcccatcta caccaacgtg
     5101 acctatccca ttacggtcaa tccgccgttt gttcccacgg agaatccgac gggttgttac
     5161 tcgctcacat ttaatgttga tgaaagctgg ctacaggaag gccagacgcg aattattttt
     5221 gatggcgttg gaattagctt atcgactgca cggtgcacca atgcttctgg cgtcaggcag
     5281 ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc
     5341 aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg ttctggcaaa
     5401 tattctgaaa tgagctg
//
