LOCUS       sequence-420926-        6726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid in E. coli BL21(DE3) , which can be used for 
            expression and purification to get protein LolT_C41S.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6726)
  AUTHORS   Gao J, Zhou C, Hai Y
  TITLE     Stereoselective Biocatalytic alpha-Deuteration of L-Amino Acids by a
            Pyridoxal 5'-Phosphate-Dependent Mannich Cyclase.
  JOURNAL   Chembiochem. 2023 Oct 1:e202300561. doi: 10.1002/cbic.202300561.
  PUBMED    37779345
REFERENCE   2  (bases 1 to 6726)
  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: 
            "Chembiochem. 2023 Oct 1:e202300561. doi: 10.1002/cbic.202300561."
COMMENT     Sequence Label: pET28a_LolT_C41S
FEATURES             Location/Qualifiers
     source          1..6726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1604..1621)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1631..1648)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1667..1689
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     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)."
     primer_bind     complement(1728..1747)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1729..1747)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1831..1850)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2056..2133
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2134..3216
                     /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(2153..2172)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4025..4216
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4210..4232)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4318..4457
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4373..4392
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4472..4489)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4643..5231)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4723..4742)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5342..5361)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5353..6168
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5353..6168
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5426..5445)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6260..6715)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6397..6418)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6609..6628
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagctcgagt tagtgcttac
      181 tgatgacgtg tcccttcttt cctattgtct cgcatatttc cttcaacacg ttgccgatcc
      241 actcgaagtc tgacgtgtcc aaatatattt gggcgcaaag gcgtacccaa atataaccac
      301 cgtgatcgat caatgggagc caagttttgt gtgtttctgc caaggtcatc gagaggtaat
      361 gcacggccgg acctacctct tcgtcactaa gcgggctgta aggggtacca gaatgcggtg
      421 gcggctttaa ggatgagctc acagcgagag gtaagcgcac tgtggctatt ccgcagtcgc
      481 gcatctccga aggtgtgcgt tggctcttat actctccggc cggcttctcg tctaatactt
      541 cagtgcccaa tattgccgct actctatcgc cgccctcttt ggctaacacg cgcaaatact
      601 ggtagatagc ctcttcacca ccgcacactt cccgccggaa cttaagcgct gtaggaatac
      661 acatgtgcgg catattatca gacgttgcga cgtatgcgaa gatggtctca aagtctgtag
      721 ctgttgaacc ggatgaccca ccaccagact gcttagacca caggggcagg gccggcttgc
      781 cgtcccgggg tatgaaaccg aatgacggcg gaatagttga tctaatataa tgttgatttc
      841 gctctggaac ataaagggct gcgcaaggcc gtggcacgaa aagccatttg tgacaatcca
      901 tgatgaagaa gtcaggctga agaacctcaa gatttaagga aaactgtccg atagagtgag
      961 cgccgtccac caaactcagt actccctctc tttggcatgc ctgtaccagg ctctcaaaag
     1021 gcatgcgtat ggcggggatc gaaacaatcg tctcaaggac agcaaggcga gggtgcaggc
     1081 cctcggcttt aatctgtgcc accatcccct cgaatcttgc gacgatgtct tctccgcgca
     1141 tcggtaaatc gaccgttact ttgcgtgttt ggaaaggttg agtctctgcc atacttgcaa
     1201 tagcgttgtc aatagctccg tatgtagttg aaaacgtaat gatcacgtca tccttatcga
     1261 aagggatgtt atgcaaaact gtgtagattc cagtcgtagc gttgctgatg aaaacgcact
     1321 cgctcactct tgcgtgcagg agctgtgcaa cttcccgtcg cgcttcctgt ataacgggtc
     1381 cttgtttcac ttcgctgaaa taatcaggtt gtgcttcaag gtcgtctaaa tatctacgcc
     1441 attggtcgcg tacggtcttt ggccagctgc cacttgacgg ggcatttaaa ttccggtatt
     1501 ccggattcat acagaaatgc ttaagcatcg gctttccgaa tggcgtagtt ggacgtttcc
     1561 aaccttcatt ttccgacagt ggaagggcgg gcatatgcat atggctgccg cgcggcacca
     1621 ggccgctgct gtgatgatga tgatgatggc tgctgcccat ggtatatctc cttcttaaag
     1681 ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt
     1741 cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc gtggccggca
     1801 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag
     1861 atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc
     1921 ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg
     1981 tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag
     2041 agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag
     2101 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
     2161 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
     2221 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
     2281 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
     2341 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
     2401 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
     2461 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
     2521 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
     2581 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
     2641 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
     2701 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
     2761 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
     2821 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
     2881 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
     2941 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
     3001 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     3061 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     3121 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     3181 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
     3241 gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag
     3301 ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat
     3361 catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt
     3421 tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct
     3481 cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat
     3541 cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct
     3601 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg
     3661 aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt
     3721 ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat
     3781 ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg
     3841 ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt
     3901 accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat
     3961 cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc
     4021 atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac
     4081 attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga
     4141 atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga
     4201 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     4261 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     4321 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     4381 gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga
     4441 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4501 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4561 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4621 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4681 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4741 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4801 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4861 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4921 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4981 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5041 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     5101 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5161 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5221 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5281 actcacgtta agggattttg gtcatgaaca ataaaactgt ctgcttacat aaacagtaat
     5341 acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat
     5401 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     5461 ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     5521 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     5581 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     5641 ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca
     5701 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     5761 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     5821 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     5881 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     5941 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     6001 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     6061 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     6121 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga attaattcat
     6181 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6241 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     6301 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     6361 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     6421 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     6481 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     6541 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     6601 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     6661 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     6721 tcgcca
//
