LOCUS       sequence-271593-        6332 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SaDAH::G226D variant with N-terminal 8xHis-tag in BL21 E. 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6332)
  AUTHORS   Kim CY, Mitchell AJ, Glinkerman CM, Li FS, Pluskal T, Weng JK
  TITLE     The chloroalkaloid (-)-acutumine is biosynthesized via a Fe(II)- and
            2-oxoglutarate-dependent halogenase in Menispermaceae plants.
  JOURNAL   Nat Commun. 2020 Apr 20;11(1):1867. doi: 10.1038/s41467-020-15777-w.
  PUBMED    32313070
REFERENCE   2  (bases 1 to 6332)
  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: "Nat 
            Commun."; date: "2020-04-20"; volume: "11(1):1867. doi"; pages: " 
            10.1038/s41467-020-15777-w"
COMMENT     Sequence Label: pHIs8-4b-SaDAH::G226D
FEATURES             Location/Qualifiers
     source          1..6332
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     156..173
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    188..330
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(256..275)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     416..438
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(432..623)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1432..2514)
                     /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     2476..2495
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2515..2592)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2798..2817
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2901..2920
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2901..2919
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2920..2944
                     /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             2959..2981
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2994..3017
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3024..3044
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(4163..4181)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4177..4224
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4261..4716
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4348..4367)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4558..4579
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4809..5624)
                     /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             complement(4809..5624)
                     /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     5532..5551
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5616..5635
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(5746..6332,1..2)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6235..6254
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
       61 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
      121 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
      181 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
      241 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
      301 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
      361 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
      421 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
      481 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
      541 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
      601 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
      661 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
      721 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
      781 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
      841 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
      901 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
      961 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     1021 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     1081 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     1141 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     1201 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     1261 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     1321 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     1381 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     1441 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     1501 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     1561 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     1621 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     1681 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     1741 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     1801 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     1861 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     1921 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     1981 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     2041 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     2101 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     2161 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     2221 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     2281 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     2341 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     2401 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     2461 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     2521 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     2581 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     2641 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     2701 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     2761 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     2821 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     2881 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     2941 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg aaacaccacc
     3001 accaccacca ccaccacggt ggtgaaaact tgtacttcca ggcccatggc atggaagtcg
     3061 agaaggtact ccaaccaatc agcaagccag gggaatcggg ctatgatcac ttcatgccgg
     3121 aggactttat tcgtccagaa gacgaacagc cagaattaac aacctttaag ggcccaaccc
     3181 cggacattcc ggttattgac ttgtcggaac cgaacgaaga aaagttggtt cgtgccttgg
     3241 tatcagcatc cgaggagtgg ggcatctttc aagttgttaa tcacgggatt ccgacagatg
     3301 ttattgacaa atttatgcgg gttgggcggg agtttttcga actgccgcag gaggaaaaga
     3361 tggcatatgc gcggccacca ggggcgacct cactggaggg ctacgagacc cgtttcgaac
     3421 gggattatgt gggcaaaaag gcgtgggctg acctcctgtt tcacaacgtc tggccaccaa
     3481 gcattgttaa ctattccttc tggccgaaaa atcctccttc ataccgcgaa gccacagaag
     3541 aatatgctaa acatattccg attgtcgcgg ataagctttt taaaatcctt agtctgggtc
     3601 tcggccttga ggggaacaca attaaagaag gtttaggggg cgagaagatg gaatttctta
     3661 tgaaaatcaa ttattaccct ccttgtccgc gccctgattt ggctcttggt gtagtccctc
     3721 atacagacta tcctgcaatg acaattttga tccctacaga tgtaccgggc cttcaagtgt
     3781 tcaaggatga cctgtggtat gacgccaagt atattccata cgccctcgtg gtcaatattg
     3841 ctgatcaaat cgagattctt agcaatggta agtacaagag tgtactccac cgcgcgaaag
     3901 tcaataaaga gaaggttcgc atgtcctggc cgattttttg tacgcctccg gcagatatgg
     3961 ttattggccc tatccctgaa ttgatcaacg aagagaaccc ggctcggtac aagtacattg
     4021 aatacaaaga ttatgtccag ttgaaactta aaaataagtc tgcagccttc gagggcccga
     4081 acgtagcgaa agtgacctca aaatagccat ggcgatccgg ctgctaacaa agcccgaaag
     4141 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     4201 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     4261 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4321 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4381 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4441 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     4501 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4561 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     4621 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     4681 acgcgaattt taacaaaata ttaacgttta caatttcagg tggcactttt cggggaaatg
     4741 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4801 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     4861 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     4921 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     4981 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     5041 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     5101 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     5161 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     5221 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     5281 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     5341 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     5401 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     5461 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     5521 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     5581 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     5641 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     5701 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5761 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5821 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5881 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5941 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6001 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6061 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6121 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6181 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6241 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6301 gtgatgctcg tcaggggggc ggagcctatg ga
//
