LOCUS       sequence-366803-        8487 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of ChE1 v4.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8487)
  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 8487)
  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: pET-ChE1 v4.5
FEATURES             Location/Qualifiers
     source          1..8487
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3140..3169
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             3219..3236
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3289..3307)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3303..3350
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3387..3842
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3474..3493)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3684..3705
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3868..3972
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3973..4041,4042..4833)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4191..4210)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5004..5592
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5493..5512
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5746..5763
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5778..5917
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5843..5862)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6003..6025
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6019..6210)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7019..8101)
                     /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     8063..8082
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8102..8179)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8385..8404
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat ggccaagaac ggcagtgaag cagacataga
      121 cgagggcctt tactcccggc agctgtatgt gttgggccat gaggcaatga agcggctcca
      181 gacatccagt gtcctggtat caggcctgcg gggcctgggc gtggagatcg ctaagaacat
      241 catccttggt ggggtcaagg ctgttaccct acatgaccag ggcactgccc agtgggctga
      301 tctttcctcc cagttctacc tgcgggagga ggacatcggt aaaaaccggg ccgaggtatc
      361 acagccccgc ctcgctgagc tcaacagcta tgtgcctgtc actgcctaca ctggacccct
      421 cgttgaggac ttccttagtg gtttccaggt ggtggtgctc accaacaccc ccctggagga
      481 ccagctgcga gtgggtgagt tctgtcacaa ccgtggcatc aagctggtgg tggcagacac
      541 gcggggcctg tttgggcaac tcttctgtga ctttggagag gaaatgatcc tcacagattc
      601 caatggggag cagccactca gtgctatggt ttctatggtt accaaggaca accccggtgt
      661 ggttacctgc ctggatgagg cccgacacgg gtttgagagc ggggactttg tctccttttc
      721 agaagtacag ggcatggttg aactcaacgg aaatcagccc atggagatca aagtcctggg
      781 tccttatacc tttagcatct gtgacacctc caacttctcc gactacatcc gtggaggcat
      841 cgtcagtcag gtcaaagtac ctaagaagat tagctttaaa tccttggtgg cctcactggc
      901 agaacctgac tttgtggtga cggacttcgc caagttttct cgccctgccc agctgcacat
      961 tggcttccag gccctgcacc agttctgtgc tcagcatggc cggccacccc ggccccgcaa
     1021 tgaggaggat gcagcagaac tggtagcctt agcacaggct gtgaatgctc gagccctgcc
     1081 agcagtgcag caaaataacc tggacgagga cctcatccgg aagctggcat atgtggctac
     1141 tggggatctg gcacccataa acgccttcat tgggggcctg gctgcccagg aagtcatgaa
     1201 ggcctgctcc gggaagttca tgcccatcat gcagtggcta tactttgatg cccttgagtg
     1261 tctccctgtc gacaaagagg tcctcacaga agacaagtgc ctccagcgcc agaaccgtta
     1321 tgacgggcaa gtggctgtgt ttggctcaga cctgcaagag aagctgggca agcagaagta
     1381 tttcctggtg ggtgcggggg ccattggctg tgagctgctc aagaactttg ccatgattgg
     1441 gctgggctgc ggggagggtg gagaaatcat cgttacagac atggacacca ttgagaagtc
     1501 aaatctgaat cgacagtttc ttttccggcc ctgggatgtc acgaagttaa agtctgacac
     1561 ggctgctgca gctgtgcgcc aaatgaatcc acatatccgg gtgacaagcc accagaaccg
     1621 tgtgggtcct gacacggagc gcatctatga tgacgatttt ttccaaaacc tagatggcgt
     1681 ggccaatgcc ctggacaacg tggatgcccg catgtatatg gaccgccgct gtgtctacta
     1741 ccggaagcca ctgctggagt caggcacact gggtaccaaa ggcaatgtgc aggtggtgat
     1801 ccccttcctg acagagtcgt acagttccag ccaggaccca cctgagaagt ccatccccat
     1861 ctgtaccctg aagaacttcc ctaatgccat cgagcacacc ctgcagtggg ctcgggatga
     1921 gtttgaaggc ctcttcaagc agccagcaga aaatgtcaac cagtacctca cagaccccaa
     1981 gtttgtggag cgaacactgc ggctgacagg cactcaaccc ttggaggtgc tggaggccgt
     2041 gcagcgcagc ctggtgctgc agcgaccaca gacctgggct gactgcgtga cctgggcctg
     2101 ccaccactgg cacacccagt actcgaacaa catccggcag ctgctgcact acatccctcc
     2161 tgaccagctc acaagctcgg gagcgccgtt ctggtctggg cccaaacgct gtccacaccc
     2221 gctcaccttt gatgtcaaca atcccctgca tctggactat gtgatggctg ctgccaacct
     2281 gtttgcccag acttacgggc tgacaggctc tcaggaccga gctgctgtgt ccacattcct
     2341 gcagtctgtg caggtccccg aatttacccc caagtctggc gtcaagatcc atgtttctga
     2401 ccaggagctg cagagcgcca atgcctctgt tgatgacagt cgtcttgagg agctcaaagc
     2461 cactctgccc agcccagaca agctccctgg attcaagatg taccccattg actttgagaa
     2521 ggatgaagac agcaactttc atatggattt catcgaggct gcatccaacc tccgggcaga
     2581 aaactatgac attccttctg cagaccggca caagagcaag ctgattgcag ggaagatcat
     2641 ccccgccatt gccacgacct cagcagccgt ggttggcctt gtgtgtctgg aactgtataa
     2701 ggttgtgcag gggcaccgac agcttgactc ctacaagaat ggtttcctca acttggccct
     2761 gcctttcttt ggtttctctg aaccccttgc cgcaccacgt caccagtact ataaccaaga
     2821 gtggacattg tgggatcgcg tgactgtgcg gctgaatgta cataaagtga ctgttctcac
     2881 cttacaagac aagatagtga aagaaaaatt tgctatggta gcaccagatg tccaaattga
     2941 agatgggaaa ggaacaatcc taatatcttc cgaagaggga gagacggaag ctaataatca
     3001 caagaagttg tcagaatttg gaattagaaa tggcagccgg cttcaagcag atgacttcct
     3061 ccaggactat actttattga tcaacatcct tcatagtgaa gaccttggaa aggacgttga
     3121 atttgaagtt gttggtgatc atcaccatca ccatcaccat caccatcact aaggatccga
     3181 attcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac caccactgag
     3241 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     3301 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     3361 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     3421 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3481 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3541 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3601 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3661 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3721 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3781 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     3841 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3901 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3961 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4021 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4081 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4141 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4201 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4261 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4321 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4381 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4441 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4501 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     4561 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     4621 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     4681 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     4741 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     4801 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     4861 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4921 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4981 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5041 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5101 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     5161 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5221 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5281 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5341 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5401 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5461 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5521 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5581 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5641 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     5701 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     5761 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     5821 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     5881 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     5941 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     6001 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     6061 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     6121 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     6181 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     6241 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     6301 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     6361 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     6421 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     6481 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     6541 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     6601 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     6661 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     6721 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     6781 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     6841 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     6901 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     6961 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     7021 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     7081 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     7141 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     7201 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     7261 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     7321 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     7381 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     7441 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     7501 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     7561 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     7621 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     7681 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     7741 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     7801 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     7861 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     7921 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     7981 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     8041 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     8101 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     8161 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     8221 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     8281 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     8341 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     8401 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     8461 aggatcgaga tctcgatccc gcgaaat
//
