LOCUS       sequence-305905-        6707 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express LanCL2-C321A/C367A 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-C321A/C367A-C-Ter-His
FEATURES             Location/Qualifiers
     source          1..6707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(48..636)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(128..147)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        717..809
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(810..878,879..1670)
                     /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     complement(1028..1047)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1785..2240)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1922..1943)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2134..2153
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2277..2324)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2320..2338
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2376..2420)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     protein_bind    2529..2553
                     /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)."
     promoter        complement(2554..2572)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2638..2655)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4012..4034
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4049..4073
                     /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)."
     promoter        complement(4074..4092)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4180..4199)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4405..4482
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4483..5565
                     /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(4502..4521)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6137..6328
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6322..6344)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6430..6569
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6485..6504
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6584..6601)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
       61 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      121 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      181 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      241 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      301 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
      361 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
      421 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
      481 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
      541 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
      601 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
      661 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gacctatttg
      721 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      781 gcttcaatca tgattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
      841 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
      901 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
      961 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1021 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1081 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1141 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1201 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     1261 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     1321 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     1381 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     1441 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     1501 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     1561 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     1621 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     1681 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     1741 aggtgaagat cctttttgat aatctcatgc catcgtgccg ccagaaattg taaacgttaa
     1801 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     1861 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     1921 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     1981 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     2041 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     2101 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     2161 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     2221 tgcgccgcta cagggcgcgt cccattcgcc aatccggata tagttcctcc tttcagcaaa
     2281 aaacccctca agacccgttt agaggcccca aggggttatg ctagttattg ctcagcggtg
     2341 gcagcagcct aggttaatta agctgcgcta gtagacgagt ccatgtgctg gcgttcaaat
     2401 ttcgcagcag cggtttcttt accagactcg agggtaccga cgtcagcgat cgcgtggccg
     2461 gccgatatcc aattgagatc tgccatatgt atatctcctt cttatactta actaatatac
     2521 taagatgggg aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgat
     2581 tatgcggccg tgtacaatac gattactttc tgttcgactt aagcattatg cggccgcgtg
     2641 gtgatgatgg tgatgatccc tcttcgaaga gtcaagttca aatgctggaa accgtgatgt
     2701 ctctggtccc aggacatcag agagaaagtg aatagcgcca gccatgcctt caaagagcga
     2761 atagggtctg tcaggaatgc ggcacccgtg tgctccgtaa tctagacacc actctgcaaa
     2821 cttgcaagct cggtagaggt acttcttatc ctgcgtgaga cggtaaaggg acaggaagga
     2881 atagccgttg ccagcagtcc catgggctat cccgtagccc ttccgcagca aacctcgctg
     2941 ccaaatcaca tcgctacact ccatggcctc tttcaagtac ttctcctcct taaagacctt
     3001 gtacgcctgc atgagcatgt ggatgacccc cggggcgccg tgggcccagt gcaccagccg
     3061 gtctgtttca ttgcttaatg atgatgggta attcccagat cggaattttt tgtggcgcac
     3121 ataatcaata ctgggtttca ccatttctgt caaggtttct tggtccactt ttgctgccgg
     3181 ctgcattaac atatagtaaa ttccagccat gccatgggct gctccaacgt actgcttccg
     3241 gtgccactgg tacaacagcg ggcagcgctc cgtttttctt tcttcccttg acaaagtctt
     3301 acccgattca ataatagcat tgactacctc tttaatagct gactcacaca cggtgcctgg
     3361 acctatctct gtgttcaggt acagtaaggc atacagataa cctgcccgtc cataaagcag
     3421 ctcatcagga aggtctgatt cttggcagac aaccgatctc tggagctgca aaagttttgt
     3481 gacacattcc tgggactcac agtcacttct gagtttgtga taaatcacag ctccaacagc
     3541 cagggggcca gcatccccac agaggaaggt gaccctgcgg ccattcagat tccgaagtgt
     3601 tctttttacg taatccaggg atcggagcag gtaggtttgg tcacatgtga cccggtacaa
     3661 ctgcaggtac aaaagggcta tgcctgtcca gccagtataa gcagagcagt catggggatc
     3721 agctgtcttc agcccttctt ccatttgctg cagaagatct ttaattttgg tctggatccg
     3781 tcttatgaaa ttatgaatga tcttcccgtc ctgatgaaaa gggaggccgg gctcatccgg
     3841 ggtggccggg ggacgaacac agcctgtctc ttcggccgct ccggaggcga gcagcgcccc
     3901 ggcggcggcc tcgtagtccg ggaaggggtt gacgaacgcc cgttcctcca tttctgcctc
     3961 ccctcccagg tggagcttca gcctctttga catggtctcg cccatggtat atctccttct
     4021 taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag
     4081 tgagtcgtat taatttcgcg ggatcgagat cgatctcgat cctctacgcc ggacgcatcg
     4141 tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg
     4201 atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg
     4261 tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg
     4321 cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc
     4381 ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg
     4441 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta
     4501 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
     4561 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
     4621 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
     4681 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
     4741 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
     4801 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
     4861 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
     4921 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
     4981 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     5041 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     5101 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     5161 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
     5221 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     5281 gacatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
     5341 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     5401 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     5461 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     5521 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     5581 tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa
     5641 cccagtcagc tccttccggt gggcgcgggg catgactagc atgatcgtgc tcctgtcgtt
     5701 gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg
     5761 cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat
     5821 ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat
     5881 tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt
     5941 attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac
     6001 cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg
     6061 tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc
     6121 ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat
     6181 cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc
     6241 agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg
     6301 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     6361 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     6421 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac
     6481 tatgcggcat cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca
     6541 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     6601 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6661 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaa
//
