LOCUS       sequence-275079-        6328 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 6328)
  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 6328)
  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..6328
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     156..173
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    188..327
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(253..272)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     413..435
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(429..620)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1429..2511)
                     /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     2473..2492
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2512..2589)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2795..2814
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2898..2917
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2898..2916
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2917..2941
                     /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             2956..2978
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2991..3014
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3021..3041
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQA"
     primer_bind     complement(4160..4178)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4174..4221
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4258..4713
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4345..4364)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4555..4576
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4805..5620)
                     /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(4805..5620)
                     /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     5528..5547
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5612..5631
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(5742..6328,1..2)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6231..6250
                     /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 caccgcaatg
      241 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
      301 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
      361 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
      421 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
      481 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
      541 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
      601 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
      661 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
      721 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
      781 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
      841 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
      901 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
      961 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     1021 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     1081 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     1141 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     1201 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     1261 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     1321 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     1381 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     1441 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     1501 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     1561 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     1621 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     1681 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     1741 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     1801 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     1861 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     1921 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     1981 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     2041 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     2101 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     2161 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     2221 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     2281 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     2341 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     2401 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     2461 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     2521 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     2581 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     2641 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     2701 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     2761 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     2821 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     2881 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     2941 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgaaa caccaccacc
     3001 accaccacca ccacggtggt gaaaacttgt acttccaggc ccatggcatg gaagtcgaga
     3061 aggtactcca accaatcagc aagccagggg aatcgggcta tgatcacttc atgccggagg
     3121 actttattcg tccagaagac gaacagccag aattaacaac ctttaagggc ccaaccccgg
     3181 acattccggt tattgacttg tcggaaccga acgaagaaaa gttggttcgt gccttggtat
     3241 cagcatccga ggagtggggc atctttcaag ttgttaatca cgggattccg acagatgtta
     3301 ttgacaaatt tatgcgggtt gggcgggagt ttttcgaact gccgcaggag gaaaagatgg
     3361 catatgcgcg gccaccaggg gcgacctcac tggagggcta cgagacccgt ttcgaacggg
     3421 attatgtggg caaaaaggcg tgggctgacc tcctgtttca caacgtctgg ccaccaagca
     3481 ttgttaacta ttccttctgg ccgaaaaatc ctccttcata ccgcgaagcc acagaagaat
     3541 atgctaaaca tattccgatt gtcgcggata agctttttaa aatccttagt ctgggtctcg
     3601 gccttgaggg gaacacaatt aaagaaggtt tagggggcga gaagatggaa tttcttatga
     3661 aaatcaatta ttaccctcct tgtccgcgcc ctgatttggc tcttggtgta gtccctcata
     3721 cagactatcc tgcaatgaca attttgatcc ctacagatgt accgggcctt caagtgttca
     3781 aggatgacct gtggtatgac gccaagtata ttccatacgc cctcgtggtc aatattgctg
     3841 atcaaatcga gattcttagc aatggtaagt acaagagtgt actccaccgc gcgaaagtca
     3901 ataaagagaa ggttcgcatg tcctggccga ttttttgtac gcctccggca gatatggtta
     3961 ttggccctat ccctgaattg atcaacgaag agaacccggc tcggtacaag tacattgaat
     4021 acaaagatta tgtccagttg aaacttaaaa ataagtctgc agccttcgag ggcccgaacg
     4081 tagcgaaagt gacctcaaaa tagccatggc gatccggctg ctaacaaagc ccgaaaggaa
     4141 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     4201 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     4261 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4321 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4381 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4441 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     4501 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4561 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     4621 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4681 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     4741 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     4801 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     4861 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     4921 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     4981 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     5041 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     5101 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     5161 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     5221 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     5281 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     5341 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     5401 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     5461 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     5521 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     5581 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     5641 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     5701 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5761 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5821 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5881 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5941 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6001 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6061 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6121 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6181 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6241 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6301 tgctcgtcag gggggcggag cctatgga
//
