LOCUS       sequence-305788-        6902 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express LanCL1-C322A in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6902)
  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 6902)
  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: pET-15b-LanCL1-C322A
FEATURES             Location/Qualifiers
     source          1..6902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     74..93
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     177..196
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        177..195
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    196..220
                     /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             235..257
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             276..293
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             303..320
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1579..1597)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1593..1640
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1815..1820,1821..1837,1838..1843))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1870..1888)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1956..2060
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2061..2129,2130..2921)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2279..2298)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3092..3680
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3581..3600
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3834..3851
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3866..4005
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3931..3950)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4091..4113
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4107..4298)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5610..6692)
                     /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     6654..6673
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6693..6770)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
       61 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
      121 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
      181 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
      241 taactttaag aaggagatat accatgggca gcagccatca tcatcatcat cacagcagcg
      301 gcctggtgcc gcgcggcagc catatggctc aaagggcctt cccgaatcct tatgctgatt
      361 ataacaaatc cctggccgaa ggctactttg atgctgccgg gaggctgact cctgagttct
      421 cacaacgctt gaccaataag attcgggagc ttcttcagca aatggagaga ggcctgaaat
      481 cagcagaccc tcgggatggc accggttaca ctggctgggc aggtattgct gtgctttact
      541 tacatcttta tgatgtattt ggggaccctg cctacctaca gttagcacat ggctatgtaa
      601 agcaaagtct gaactgctta accaagcgct ccatcacctt cctttgtggg gatgcaggcc
      661 ccctggcagt ggccgctgtg ctatatcaca agatgaacaa tgagaagcag gcagaagatt
      721 gcatcacacg gctaattcac ctaaataaga ttgatcctca tgctccaaat gaaatgctct
      781 atgggcgaat aggctacatc tatgctcttc tttttgtcaa taagaacttt ggagtggaaa
      841 agattcctca aagccatatt cagcagattt gtgaaacaat tttaacctct ggagaaaacc
      901 tagctaggaa gagaaacttc acggcaaagt ctccactgat gtatgaatgg taccaggaat
      961 attatgtagg ggctgctcat ggcctggctg gaatttatta ctacctgatg cagcccagcc
     1021 ttcaagtgag ccaagggaag ttacatagtt tggtcaagcc cagtgtagac tacgtctgcc
     1081 agctgaaatt cccttctggc aattaccctc catgtatagg tgataatcga gatctgcttg
     1141 tccattggtg ccatggcgcc cctggggtaa tctacatgct catccaggcc tataaggtat
     1201 tcagagagga aaagtatctc tgtgatgcct atcagtgtgc tgatgtgatc tggcaatatg
     1261 ggttgctgaa gaagggatat gggctggccc acggttctgc agggaatgcc tatgccttcc
     1321 tgacactcta caacctcaca caggacatga agtacctgta tagggcctgt aagtttgctg
     1381 aatggtgctt agagtatgga gaacatggat gcagaacacc agacacccct ttctctctct
     1441 ttgaaggaat ggctggaaca atatatttcc tggctgacct gctagtcccc acaaaagcca
     1501 ggttccctgc atttgaactc tgactcgagg atccggctgc taacaaagcc cgaaaggaag
     1561 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1621 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatccc gcaagaggcc
     1681 cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat
     1741 gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg
     1801 tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag aattcttgaa
     1861 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     1921 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1981 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2041 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2101 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2161 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     2221 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2281 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     2341 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2401 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2461 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2521 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2581 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     2641 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2701 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2761 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2821 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2881 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2941 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3001 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3061 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3121 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3181 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     3241 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3301 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3361 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3421 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3481 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3541 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3601 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3661 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3721 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3781 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3841 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     3901 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3961 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4021 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4081 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4141 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     4201 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     4261 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     4321 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     4381 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     4441 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     4501 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     4561 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     4621 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     4681 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     4741 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
     4801 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
     4861 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
     4921 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
     4981 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
     5041 ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa
     5101 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
     5161 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
     5221 ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc
     5281 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     5341 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     5401 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     5461 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     5521 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     5581 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5641 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5701 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     5761 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     5821 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     5881 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     5941 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     6001 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     6061 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     6121 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     6181 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     6241 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     6301 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     6361 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     6421 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     6481 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     6541 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     6601 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     6661 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     6721 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     6781 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     6841 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     6901 ca
//
