LOCUS       sequence-430560-        6622 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HisSUMO-RdhF3 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6622)
  AUTHORS   Ng TL, Silver PA
  TITLE     Sustainable B(12)-Dependent Dehalogenation of Organohalides in E. 
            coli.
  JOURNAL   ACS Chem Biol. 2024 Feb 16;19(2):380-391. doi: 
            10.1021/acschembio.3c00585. Epub 2024 Jan 22.
  PUBMED    38254247
REFERENCE   2  (bases 1 to 6622)
  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"; doi: 
            "10.1021/acschembio.3c00585"; journalName: "ACS Chem Biol"; date: 
            "2024-02-16- 16"; volume: "19"; issue: "2"; pages: "380-391"
COMMENT     Sequence Label: pCDB179-RdhF3
FEATURES             Location/Qualifiers
     source          1..6622
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(55..73)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      69..116
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      153..608
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(240..259)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     450..471
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(700..1515)
                     /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(700..1515)
                     /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     1423..1442
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1507..1526
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1637..2225
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2126..2145
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2379..2396
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2411..2550
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2476..2495)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2636..2658
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2652..2843)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3652..4734)
                     /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     4696..4715
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4735..4812)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5018..5037
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5125..5144
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5125..5143
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5148..5164
                     /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             5183..5205
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5219..5248
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
ORIGIN
        1 tcgaggatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
       61 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
      121 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
      181 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
      241 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
      301 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
      361 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
      421 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
      481 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
      541 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
      601 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
      661 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
      721 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
      781 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
      841 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
      901 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
      961 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     1021 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     1081 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     1141 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     1201 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     1261 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     1321 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     1381 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     1441 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     1501 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     1561 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1621 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1681 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1741 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     1801 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1861 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1921 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1981 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2041 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2101 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2161 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2221 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2281 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2341 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2401 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2461 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     2521 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     2581 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     2641 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     2701 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     2761 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     2821 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     2881 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     2941 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     3001 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     3061 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     3121 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     3181 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     3241 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     3301 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     3361 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     3421 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     3481 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     3541 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     3601 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     3661 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3721 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     3781 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     3841 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     3901 gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc
     3961 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     4021 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     4081 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     4141 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     4201 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     4261 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     4321 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     4381 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     4441 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     4501 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     4561 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     4621 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     4681 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     4741 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     4801 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     4861 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     4921 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     4981 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     5041 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     5101 agatcgatct cgatcccgcg aaattaatac gactcactat agggaaattg tgagcggata
     5161 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac atatgggtca
     5221 ccaccaccac caccaccacc accaccacgg tagcctgcaa gattcggaag tcaaccaaga
     5281 agcaaagccg gaagtcaagc cggaagtgaa accggaaacc catattaacc tgaaagttag
     5341 tgacggcagc tctgaaattt tctttaagat caaaaagacc acgccgctgc gtcgcctgat
     5401 ggaagcgttt gccaaacgtc agggcaagga aatggatagc ctgcgtttcc tgtatgacgg
     5461 tattcgcatc caggcagatc aagcgccgga agacctggac atggaagaca acgacatcat
     5521 tgaagcccac cgtgaacaga ttggcggcat gaattctaat cgcacctcta aggcaccccg
     5581 tgtaaaagtt aaacgtgtgg aaaaaccgcc atacgtaatt gatcgctttg agctccgtag
     5641 atttgaccgt aaaaatacaa ttttcgatcg agttatgtgg gattcttctt ggaaaggcta
     5701 caagcgtatg tacgacgaaa aggtcctgga taccgtggca aatggtaagc ctggctactc
     5761 tcgtatagac tttgccctgg cctatgccag ctggattgtt catgacgcgt ttgagggcgg
     5821 gttcagctgg aaaaaaatca aactctatgg tacaccagcc gccacaatcg gtattgattg
     5881 gacgaagaca aaatacgaag ttgatgatcc atgcaaaatg agtaccttca ccaagcgtgc
     5941 agctaagtta tttggtgcat ctctcgtggg tatttgcaag ctgaaccggg attggctgta
     6001 tgccgacgtc aacattccgg aaaagttcga gaatgtgatc gtcatggcgg ttgaaatgga
     6061 cgcagatggc attgccacct ccccagctgc tccggcagcg gctgctaccg gtgtaggtta
     6121 ctccaaaatg gcgtttatac tggcctgtat gggtgagttc atccgtaatt taggctacga
     6181 agcaatccag tgcggaaatg acacggcatt atcgataccg ttggcgatag atgcaggtct
     6241 ggggcaactt gggcgtaacg gtttgttgat aacaccgcag tatggccctc gcgtccgttt
     6301 ctgcaaaata tttacggact tgcctttggg tccggataaa cccattaaat ttggcgtgaa
     6361 agaattttgt gagaagtgca aattgtgtgc tcaacactgc aaagctgtag caattagcat
     6421 ggataacgaa cctaactttc aggcgacttg ccgttcaaac aatcctggag ctttgaagtg
     6481 gtatgttgac gttgaaagat gctacttata ttggtgccaa aatggaatag actgcagtac
     6541 gtgtataaaa gtttgtccat acaatatagc aagtgctcgg aaagctaaag tcacacctaa
     6601 agaattttgg aatattgttt aa
//
