LOCUS       sequence-478119-        6431 bp    DNA     circular SYN 10-JUL-2025
DEFINITION  expresses N-His tagged NocO E252A (diiron halogenase variant) from 
            Nodularia sp. 06071 (mutation numbering based on amino acid position
            in native sequence), created from mutagenesis of WT plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6431)
  AUTHORS   Wang ML, Glasser NR, Nair MA, Krebs C, Martin Bollinger J Jr, 
            Balskus EP
  TITLE     Biochemical Studies of a Cyanobacterial Halogenase Support the 
            Involvement of a Dimetal Cofactor.
  JOURNAL   Biochemistry. 2025 May 20;64(10):2173-2180. doi: 
            10.1021/acs.biochem.4c00720. Epub 2025 Apr 29.
  PUBMED    40300765
REFERENCE   2  (bases 1 to 6431)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 10, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acs.biochem.4c00720"; journalName: "Biochemistry"; date: 
            "2025-05-20- 20"; volume: "64"; issue: "10"; pages: "2173-2180"
COMMENT     Sequence Label: pETDuet-solo-NHis6-NocO-E252A
FEATURES             Location/Qualifiers
     source          1..6431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1462..1480)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1476..1523
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1560..2015
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1647..1666)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1857..1878
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2130..2921,2922..2990))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2753..2772
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2991..3083)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3164..3752
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3653..3672
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3906..3923
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3938..4077
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4003..4022)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4163..4185
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4179..4370)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4942..6024)
                     /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     5986..6005
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6025..6102)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6308..6327
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6415..6431,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(6415..6431,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac ggcagcatta ctagcagtaa
      121 tacttcacca atatctcaaa ttaataaagt tatccctctc ttggaaaaga actatggaaa
      181 taaccttgac tcagactaca ctgaaaaaat ccaagaacta gatagaaact ttaactatag
      241 tgccaacagt aatgtttttt ggagtgaacc agaacactct atcctttatg gcacaccact
      301 ttatgaacaa gcttctctat ctcaaaaatt agcactcaat cacctgcatt gggtattgtt
      361 ttacaagaga gttgctgaca gtgaaactga gctaattcat tacaacacta tcacagcaga
      421 ttgtctcatg gctgcaaata gtgatcataa aatgctggtg gaactgttag ctcatgaaac
      481 ttaccaagaa cgtcaacata ttcatgcttt ttctcaaata aattataaaa ctactaaagc
      541 tttactaggt aaaaaagctc tagtgaatcc cgtttctgat gagactactg tacgggagca
      601 gaaaaaggat atatctgcca attatatatc cactgctgcc aacttgctcg caatgatgct
      661 cccaggtaaa aagcaatctc atgccgattc tgataaatat ttgtcatttc ctaccaacgg
      721 attttttaat ggattttcag gaaaatcaga tatatcaaga aaacagttct tttcagataa
      781 ttggagaagt tatccattct tggctgctaa tttttatgct gtgcgctaca tggctaatct
      841 actattgaaa aaccatgcgt ttggtataca ttgctacttc aaaaaaatgc ataaaaataa
      901 tgaatttata gcgagtccaa cagctatatc tcactatcat tttttagatg aagcttttca
      961 tacaaccact tctctatttc ttggtcgaga ctttcataaa tatttaccaa agccatcaag
     1021 atatgaaaaa tggattgcca acttagctat cttacaggca cagcacgaaa acctcaatgc
     1081 tatttctgga atatctacca accgcttttt gagtgatggt gatgtgataa tttttctgat
     1141 gaaactcttg cagagtcctg tatttgatat gtcggctcag gaagcaattc agtggattga
     1201 aaagtgccta tgttatgagc atgaaggatt tcatgtcaac ctgaaggtac ataataaact
     1261 gttatcagaa ttacgtaagt ttgcatatca actagactac ttgtggcctg taaataagga
     1321 aatgcgtgta atggcttcag gaggttcaat taagaaagcg atcaaaaata atatcaaagc
     1381 ctttaatcag ttttctatag caatttaata agcacatgga ctcgtctact agcgcagctt
     1441 aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     1501 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     1561 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1621 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1681 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1741 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     1801 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     1861 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     1921 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     1981 cgcgaatttt aacaaaatat taacgtttac aatttctggc ggcacgatgg catgagatta
     2041 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     2101 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     2161 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     2221 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     2281 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     2341 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     2401 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     2461 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     2521 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     2581 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     2641 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     2701 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     2761 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     2821 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     2881 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     2941 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     3001 tttcaatcat gattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3061 atgtatttag aaaaataaac aaataggtca tgaccaaaat cccttaacgt gagttttcgt
     3121 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3181 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3241 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3301 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3361 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3421 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3481 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3541 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3601 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3661 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3721 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3781 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3841 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3901 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3961 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     4021 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     4081 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4141 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4201 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     4261 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     4321 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     4381 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     4441 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     4501 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     4561 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     4621 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     4681 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     4741 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4801 gtcctcaacg acaggagcac gatcatgcta gtcatgcccc gcgcccaccg gaaggagctg
     4861 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     4921 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4981 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     5041 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     5101 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     5161 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     5221 gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5281 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5341 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5401 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5461 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5521 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5581 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5641 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5701 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5761 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5821 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5881 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5941 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     6001 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     6061 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     6121 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     6181 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6241 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     6301 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     6361 cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg aaattaatac
     6421 gactcactat a
//
