LOCUS       sequence-366802-        7422 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of MBP-GFP(2M IKQE).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7422)
  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 7422)
  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-MBP-GFP(2M-IKQE)
FEATURES             Location/Qualifiers
     source          1..7422
                     /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"
     CDS             840..1940
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1797..1806
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1914..1937
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1989..2000
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2004..2024
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2827..2844)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2827..2843)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2836..2858)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3022..3039)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3192..3278
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3370..3397
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3416..3507
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3508..3576,3577..4368)
                     /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(3726..3745)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4410..4923)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4605..4626)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4817..4836
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5034..5622
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5523..5542
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5776..5793
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5808..5948
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5874..5893)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6034..6056
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6050..6241)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6737..6814
                     /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(6815..7422,1..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(6834..6853)
                     /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 agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc
      901 tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc
      961 cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta
     1021 tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca
     1081 ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca
     1141 acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag
     1201 atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga
     1261 aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc
     1321 tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag
     1381 acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta
     1441 aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag
     1501 gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag
     1561 tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg
     1621 gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc
     1681 tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg
     1741 gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt attgccgcca
     1801 ccatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct
     1861 ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag
     1921 ccctgaaaga cgcgcagact aattcgagct cgaacaacaa caacaataac aataacaaca
     1981 acctcgggat cgagggaagg ggagaaaatc tttattttca aggttccggt ggatccatgc
     2041 gtaaaggcga agaattattt acgggcgtag tgcctatttt agtggaactg gatggcgatg
     2101 tgaatggaca caagtttagc gtgcgtggcg aaggtgaagg cgatgcgaca aatggcaagc
     2161 tgacgctgaa atttatctgt acgaccggta aactgccggt tccctggccg accctggtta
     2221 ccaccctgac atacggtgtg caatgcttcg cccgctatcc agatcacatg aaacagcatg
     2281 actttttcaa gagcgcgatg cccgaaggct acgttcaaga gcgtaccatc agcttcaaag
     2341 atgacggcac ttataaaacc cgcgcggaag taaagtttga aggtgataca ctggtcaatc
     2401 gcatcgaact gaagggtatt gactttaaag aagatggcaa catcttaggc cataaattag
     2461 agtataactt caactcacac aatgtgtaca ttacggcaga caaacagaag aacggtatta
     2521 aggccaactt caagattcgt cataacgtag aagatggtag cggtccgcgt gcggttatca
     2581 aacaggaatc ggaagagggc tccggctcgg tccagttagc ggatcactat caacagaata
     2641 cccctattgg cgatggtcca gttctgctgc cggataatca ctacctgagt acgcagtcag
     2701 tcctgtcgaa agacccgaat gagaaacgcg accatatggt tctgttagaa ttcgtaactg
     2761 ccgcaggcat tacccacggc atggacgagc tgtacaaata ggagtcgacc tgcaggcaag
     2821 cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     2881 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     2941 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcagcttg gctgttttgg
     3001 cggatgagat aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat
     3061 aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc
     3121 agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa
     3181 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     3241 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     3301 ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc
     3361 aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttttgt
     3421 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3481 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     3541 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     3601 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     3661 ggtaagatcc ttgagagttt tcgccccgaa gaacgtttcc caatgatgag cacttttaaa
     3721 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     3781 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     3841 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     3901 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     3961 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4021 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     4081 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4141 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4201 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     4261 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4321 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     4381 gtttactcat atatacttta gattgattta ccccggttga taatcagaaa agccccaaaa
     4441 acaggaagat tgtataagca aatatttaaa ttgtaaacgt taatattttg ttaaaattcg
     4501 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     4561 cttataaatc aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga
     4621 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     4681 atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag
     4741 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     4801 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     4861 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     4921 cgtaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4981 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5041 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5101 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5161 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     5221 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5281 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5341 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5401 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5461 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5521 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5581 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5641 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     5701 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     5761 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     5821 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     5881 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     5941 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     6001 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     6061 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgca
     6121 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     6181 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     6241 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     6301 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     6361 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     6421 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     6481 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     6541 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     6601 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     6661 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa
     6721 cgctgcccga aattccgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata
     6781 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     6841 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     6901 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     6961 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     7021 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     7081 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     7141 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     7201 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     7261 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     7321 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     7381 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aa
//
