LOCUS       sequence-305911-        6707 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express LanCL2-H264A in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6707)
  AUTHORS   Lai KY, Galan SRG, Zeng Y, Zhou TH, He C, Raj R, Riedl J, Liu S, 
            Chooi KP, Garg N, Zeng M, Jones LH, Hutchings GJ, Mohammed S, Nair 
            SK, Chen J, Davis BG, van der Donk WA
  TITLE     LanCLs add glutathione to dehydroamino acids generated at 
            phosphorylated sites in the proteome.
  JOURNAL   Cell. 2021 Apr 27. pii: S0092-8674(21)00436-0. doi: 
            10.1016/j.cell.2021.04.001.
  PUBMED    33932340
REFERENCE   2  (bases 1 to 6707)
  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: "Cell. 2021 
            Apr 27. pii: S0092-8674(21)00436-0. doi: 
            10.1016/j.cell.2021.04.001."
COMMENT     Sequence Label: pETDuet1-LanCL2-H264A-C-Ter-His
FEATURES             Location/Qualifiers
     source          1..6707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     58..77
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        165..183
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    184..208
                     /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             223..245
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1602..1619
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1685..1703
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1704..1728
                     /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             1837..1881
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1919..1937)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1933..1980
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2017..2472
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2104..2123)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2314..2335
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2587..3378,3379..3447))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3210..3229
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3448..3540)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3621..4209
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4110..4129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4363..4380
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4395..4534
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4460..4479)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4620..4642
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4636..4827)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5399..6481)
                     /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     6443..6462
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6482..6559)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
       61 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
      121 gcgtccggcg tagaggatcg agatcgatct cgatcccgcg aaattaatac gactcactat
      181 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
      241 ggagatatac catgggcgag accatgtcaa agaggctgaa gctccacctg ggaggggagg
      301 cagaaatgga ggaacgggcg ttcgtcaacc ccttcccgga ctacgaggcc gccgccgggg
      361 cgctgctcgc ctccggagcg gccgaagaga caggctgtgt tcgtcccccg gccaccccgg
      421 atgagcccgg cctccctttt catcaggacg ggaagatcat tcataatttc ataagacgga
      481 tccagaccaa aattaaagat cttctgcagc aaatggaaga agggctgaag acagctgatc
      541 cccatgactg ctctgcttat actggctgga caggcatagc ccttttgtac ctgcagttgt
      601 accgggtcac atgtgaccaa acctacctgc tccgatccct ggattacgta aaaagaacac
      661 ttcggaatct gaatggccgc agggtcacct tcctctgtgg ggatgctggc cccctggctg
      721 ttggagctgt gatttatcac aaactcagaa gtgactgtga gtcccaggaa tgtgtcacaa
      781 aacttttgca gctccagaga tcggttgtct gccaagaatc agaccttcct gatgagctgc
      841 tttatggacg ggcaggttat ctgtatgcct tactgtacct gaacacagag ataggtccag
      901 gcaccgtgtg tgagtcagct attaaagagg tagtcaatgc tattattgaa tcgggtaaga
      961 ctttgtcaag ggaagaaaga aaaacggagc gctgcccgct gttgtaccag tggcaccgga
     1021 agcagtacgt tggagcagcc gccggcatgg ctggaattta ctatatgtta atgcagccgg
     1081 cagcaaaagt ggaccaagaa accttgacag aaatggtgaa acccagtatt gattatgtgc
     1141 gccacaaaaa attccgatct gggaattacc catcatcatt aagcaatgaa acagaccggc
     1201 tggtgcactg gtgccacggc gccccggggg tcatccacat gctcatgcag gcgtacaagg
     1261 tctttaagga ggagaagtac ttgaaagagg ccatggagtg tagcgatgtg atttggcagc
     1321 gaggtttgct gcggaagggc tacgggatat gccatgggac tgctggcaac ggctattcct
     1381 tcctgtccct ttaccgtctc acgcaggata agaagtacct ctaccgagct tgcaagtttg
     1441 cagagtggtg tctagattac ggagcacacg ggtgccgcat tcctgacaga ccctattcgc
     1501 tctttgaagg catggctggc gctattcact ttctctctga tgtcctggga ccagagacat
     1561 cacggtttcc agcatttgaa cttgactctt cgaagaggga tcatcaccat catcaccacg
     1621 cggccgcata atgcttaagt cgaacagaaa gtaatcgtat tgtacacggc cgcataatcg
     1681 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc atcttagtat
     1741 attagttaag tataagaagg agatatacat atggcagatc tcaattggat atcggccggc
     1801 cacgcgatcg ctgacgtcgg taccctcgag tctggtaaag aaaccgctgc tgcgaaattt
     1861 gaacgccagc acatggactc gtctactagc gcagcttaat taacctaggc tgctgccacc
     1921 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1981 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     2041 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2101 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2161 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2221 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2281 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2341 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     2401 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2461 cgtttacaat ttctggcggc acgatggcat gagattatca aaaaggatct tcacctagat
     2521 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     2581 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     2641 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     2701 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     2761 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     2821 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     2881 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     2941 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3001 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     3061 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     3121 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     3181 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     3241 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     3301 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     3361 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     3421 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatcatgat tgaagcattt
     3481 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3541 taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3601 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3661 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3721 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3781 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3841 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3901 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3961 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4021 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4081 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4141 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4201 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4261 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4321 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4381 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4441 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4501 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4561 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4621 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4681 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     4741 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     4801 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     4861 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     4921 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     4981 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5041 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5101 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5161 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5221 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5281 catgctagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     5341 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     5401 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5461 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     5521 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     5581 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     5641 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
     5701 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     5761 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     5821 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     5881 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     5941 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     6001 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     6061 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     6121 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     6181 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     6241 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     6301 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     6361 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     6421 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     6481 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     6541 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     6601 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     6661 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccat
//
