LOCUS       sequence-305786-        6902 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express LanCL1-H219A 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-H219A
FEATURES             Location/Qualifiers
     source          1..6902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     52..71
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     155..174
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        155..173
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    174..198
                     /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             213..235
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             254..271
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             281..298
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1557..1575)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1571..1618
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1793..1798,1799..1815,1816..1821))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1848..1866)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1934..2038
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2039..2107,2108..2899)
                     /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(2257..2276)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3070..3658
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3559..3578
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3812..3829
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3844..3983
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3909..3928)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4069..4091
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4085..4276)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5588..6670)
                     /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     6632..6651
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6671..6748)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
       61 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
      121 ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg
      181 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
      241 catgggcagc agccatcatc atcatcatca cagcagcggc ctggtgccgc gcggcagcca
      301 tatggctcaa agggccttcc cgaatcctta tgctgattat aacaaatccc tggccgaagg
      361 ctactttgat gctgccggga ggctgactcc tgagttctca caacgcttga ccaataagat
      421 tcgggagctt cttcagcaaa tggagagagg cctgaaatca gcagaccctc gggatggcac
      481 cggttacact ggctgggcag gtattgctgt gctttactta catctttatg atgtatttgg
      541 ggaccctgcc tacctacagt tagcacatgg ctatgtaaag caaagtctga actgcttaac
      601 caagcgctcc atcaccttcc tttgtgggga tgcaggcccc ctggcagtgg ccgctgtgct
      661 atatcacaag atgaacaatg agaagcaggc agaagattgc atcacacggc taattcacct
      721 aaataagatt gatcctcatg ctccaaatga aatgctctat gggcgaatag gctacatcta
      781 tgctcttctt tttgtcaata agaactttgg agtggaaaag attcctcaaa gccatattca
      841 gcagatttgt gaaacaattt taacctctgg agaaaaccta gctaggaaga gaaacttcac
      901 ggcaaagtct ccactgatgt atgaatggta ccaggaatat tatgtagggg ctgctgccgg
      961 cctggctgga atttattact acctgatgca gcccagcctt caagtgagcc aagggaagtt
     1021 acatagtttg gtcaagccca gtgtagacta cgtctgccag ctgaaattcc cttctggcaa
     1081 ttaccctcca tgtataggtg ataatcgaga tctgcttgtc cattggtgcc atggcgcccc
     1141 tggggtaatc tacatgctca tccaggccta taaggtattc agagaggaaa agtatctctg
     1201 tgatgcctat cagtgtgctg atgtgatctg gcaatatggg ttgctgaaga agggatatgg
     1261 gctgtgccac ggttctgcag ggaatgccta tgccttcctg acactctaca acctcacaca
     1321 ggacatgaag tacctgtata gggcctgtaa gtttgctgaa tggtgcttag agtatggaga
     1381 acatggatgc agaacaccag acaccccttt ctctctcttt gaaggaatgg ctggaacaat
     1441 atatttcctg gctgacctgc tagtccccac aaaagccagg ttccctgcat ttgaactctg
     1501 actcgaggat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1561 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1621 gaaaggagga actatatccg gatatcccgc aagaggcccg gcagtaccgg cataaccaag
     1681 cctatgccta cagcatccag ggtgacggtg ccgaggatga cgatgagcgc attgttagat
     1741 ttcatacacg gtgcctgact gcgttagcaa tttaactgtg ataaactacc gcattaaagc
     1801 ttatcgatga taagctgtca aacatgagaa ttcttgaaga cgaaagggcc tcgtgatacg
     1861 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     1921 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     1981 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     2041 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     2101 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     2161 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     2221 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     2281 tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     2341 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     2401 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     2461 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     2521 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     2581 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     2641 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     2701 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     2761 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     2821 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     2881 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     2941 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3001 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3061 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3121 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3181 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     3241 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3301 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3361 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3421 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3481 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3541 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3601 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3661 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     3721 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     3781 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     3841 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     3901 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     3961 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     4021 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4081 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     4141 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     4201 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     4261 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     4321 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     4381 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     4441 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     4501 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     4561 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     4621 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     4681 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     4741 gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag atggcggacg
     4801 cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat
     4861 tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt
     4921 cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa ggtatagggc
     4981 ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt
     5041 gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag
     5101 ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cgggcatccc
     5161 gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa
     5221 cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc
     5281 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     5341 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     5401 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     5461 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     5521 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     5581 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5641 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     5701 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     5761 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     5821 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     5881 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     5941 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     6001 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     6061 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     6121 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     6181 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     6241 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     6301 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     6361 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     6421 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     6481 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     6541 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     6601 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     6661 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     6721 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     6781 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     6841 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     6901 ac
//
