LOCUS       sequence-366798-        7245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Asp6-MBP-HRas.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7245)
  AUTHORS   Akimoto G, Fernandes AP, Bode JW
  TITLE     Site-Specific Protein Ubiquitylation Using an Engineered, Chimeric 
            E1 Activating Enzyme and E2 SUMO Conjugating Enzyme Ubc9.
  JOURNAL   ACS Cent Sci. 2022 Feb 23;8(2):275-281. doi: 
            10.1021/acscentsci.1c01490. Epub 2022 Feb 9.
  PUBMED    35237717
REFERENCE   2  (bases 1 to 7245)
  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"; doi: 
            "10.1021/acscentsci.1c01490"; journalName: "ACS Cent Sci"; date: 
            "2022-02-23- 23"; volume: "8"; issue: "2"; pages: "275-281"
COMMENT     Sequence Label: pSub-Asp6-MBP-HRas
FEATURES             Location/Qualifiers
     source          1..7245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(718..723,724..739,740..746)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    754..770
                     /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     759..781
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     regulatory      1815..1824
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1932..1955
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2007..2018
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2022..2042
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2650..2667)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2650..2666)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2659..2681)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2845..2862)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3015..3101
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3193..3220
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3239..3330
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3331..3399,3400..4191)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3549..3568)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4233..4746)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4428..4449)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4640..4659
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4857..5445
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5346..5365
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5599..5616
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5631..5771
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5697..5716)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5857..5879
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5873..6064)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6560..6637
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             join(6638..7245,1..475)
                     /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(6657..6676)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
       61 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
      121 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
      181 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
      241 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
      301 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
      361 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
      421 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      481 acgcaattaa tgtaagttag ctcactcatt aggcacaatt ctcatgtttg acagcttatc
      541 atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg
      601 gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg
      661 ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg
      721 acaattaatc atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag
      781 gaaacagcca gtccgtttag gtgttttcac gagcacttca ccaacaagga ccatagatta
      841 tgaaaactga tgatgatgat gacgacgaag aaggtaaact ggtaatctgg attaacggcg
      901 ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat accggaatta
      961 aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg
     1021 gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg
     1081 gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat ccgtttacct
     1141 gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat
     1201 cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa gagatcccgg
     1261 cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac
     1321 cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgaaaacg
     1381 gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct
     1441 tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac tccatcgcag
     1501 aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca
     1561 acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc aagggtcaac
     1621 catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt ccgaacaaag
     1681 agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta
     1741 ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag
     1801 atccacgtat tgccgccacc atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc
     1861 cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc
     1921 gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg aacaacaaca
     1981 acaataacaa taacaacaac ctcgggatcg agggaagggg agaaaatctt tattttcaag
     2041 gttccggtgg atccatgacg gaatataagt tggtggtggt aggtgccggc ggagtgggta
     2101 agtcagcact gactattcag ctgatccaaa accactttgt agacgagtac gacccgacga
     2161 tcgaggactc ctatcgtaag caggtggtaa tcgacggcga gacgtgtttg ttggatatct
     2221 tagacacggc cggccaagag gagtactctg cgatgcgtga tcaatatatg cgcacgggcg
     2281 agggtttctt gtgcgttttc gccatcaata acactaaaag ctttgaagac atccaccaat
     2341 atcgtgaaca aattaaacgc gtgaaggata gtgatgacgt ccctatggtc cttgtgggca
     2401 ataaatgcga cctcgccgcc cgcactgttg aatcacgtca ggcccaggat ctcgcacgct
     2461 cctatggtat tccttacatc gaaacgtcgg ccaagacccg ccaaggtgtc gaggatgcct
     2521 tttacacttt ggtgcgcgag attcgccaac acaagcttat caagttagag ccgcccgacg
     2581 agtccgggcc gggatgcatg agctgcaagt gcgttctctc gtaggagtcg acctgcaggc
     2641 aagcttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     2701 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     2761 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcagc ttggctgttt
     2821 tggcggatga gataagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct
     2881 gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa
     2941 ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg
     3001 gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     3061 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
     3121 acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc
     3181 atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt
     3241 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3301 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3361 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3421 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3481 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt tcccaatgat gagcactttt
     3541 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt
     3601 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3661 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     3721 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3781 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3841 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3901 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3961 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4021 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4081 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4141 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4201 caagtttact catatatact ttagattgat ttaccccggt tgataatcag aaaagcccca
     4261 aaaacaggaa gattgtataa gcaaatattt aaattgtaaa cgttaatatt ttgttaaaat
     4321 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     4381 tcccttataa atcaaaagaa tagcccgaga tagggttgag tgttgttcca gtttggaaca
     4441 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     4501 gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttggggtcg aggtgccgta
     4561 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     4621 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     4681 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     4741 gcgcgtaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4801 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4861 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4921 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4981 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     5041 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5101 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5161 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5221 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5281 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5341 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5401 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5461 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5521 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     5581 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     5641 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     5701 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     5761 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     5821 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     5881 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     5941 gcagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     6001 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     6061 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     6121 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     6181 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     6241 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     6301 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     6361 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     6421 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     6481 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggccaggacc
     6541 caacgctgcc cgaaattccg acaccatcga atggtgcaaa acctttcgcg gtatggcatg
     6601 atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga
     6661 tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag
     6721 ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat
     6781 tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac
     6841 ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga
     6901 tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa
     6961 agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct
     7021 ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct
     7081 tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg
     7141 actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc
     7201 attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataa
//
