LOCUS       sequence-250790-        6518 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bacterial expression of His-tagged OphA-delta C6-Y63F.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6518)
  AUTHORS   Song H, van der Velden NS, Shiran SL, Bleiziffer P, Zach C, Sieber 
            R, Imani AS, Krausbeck F, Aebi M, Freeman MF, Riniker S, Kunzler M, 
            Naismith JH
  TITLE     A molecular mechanism for the enzymatic methylation of nitrogen 
            atoms within peptide bonds.
  JOURNAL   Sci Adv. 2018 Aug 24;4(8):eaat2720. doi: 10.1126/sciadv.aat2720. 
            eCollection 2018 Aug.
  PUBMED    30151425
REFERENCE   2  (bases 1 to 6518)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2018-08-24"; volume: "4(8):eaat2720. doi"; pages: " 
            10.1126/sciadv.aat2720. eCollection 2018 Aug"
COMMENT     Sequence Label: OphA-delta C6-Y63F
FEATURES             Location/Qualifiers
     source          1..6518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(559..1374)
                     /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(559..1374)
                     /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     1282..1301
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1366..1385
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1494..2082
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1983..2002
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2236..2253
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2268..2407
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2333..2352)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2493..2515
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2509..2700)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3509..4591)
                     /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     4553..4572
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4592..4669)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4875..4894
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4978..4997
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4978..4996
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4997..5021
                     /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             5036..5058
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5072..5095
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             5096..5116
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             6362..6379
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6432..6450)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6446..6493
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      541 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
      601 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
      661 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
      721 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
      781 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
      841 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
      901 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
      961 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1021 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1081 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1141 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1201 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1261 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     1321 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     1381 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     1441 gtgagttttc gttccactga gcgtcagacc gtagaaaaga tcaaaggatc ttcttgagat
     1501 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     1561 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     1621 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     1681 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     1741 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     1801 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     1861 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     1921 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     1981 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     2041 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     2101 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     2161 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     2221 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     2281 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     2341 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     2401 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     2461 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     2521 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     2581 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     2641 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     2701 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     2761 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     2821 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     2881 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     2941 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     3001 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     3061 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     3121 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     3181 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     3241 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     3301 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     3361 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     3421 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     3481 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3541 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3601 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     3661 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     3721 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     3781 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     3841 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     3901 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     3961 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     4021 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     4081 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     4141 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     4201 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     4261 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     4321 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     4381 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     4441 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     4501 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     4561 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     4621 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     4681 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     4741 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     4801 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     4861 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     4921 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     4981 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     5041 ttaactttaa gaaggagata tacatatgga gcatcatcat catcatcatc atcatgagaa
     5101 tctgtatttc cagggcactt ccactcagac caaagctggc tcactcacca tcgtcggtac
     5161 cggtatcgag agtatcggac aaatgacgct tcaggcgttg tcctacatcg aagccgccgc
     5221 gaaggttttc tactgcgtca tcgaccccgc cactgaggca ttcatcctca ccaagaacaa
     5281 gaactgcgtt gacttgtttc agtattacga caatggcaag tccagattga acacttacac
     5341 ccaaatgtca gagctcatgg tcagggaagt ccgcaagggc ctcgatgtcg tgggcgtctt
     5401 ctacggccac ccaggagtgt tcgtgaaccc gtctcaccga gctctggcta tcgccaagag
     5461 tgaaggctac cgagcgagga tgcttccagg cgtgtctgcg gaagattgtc tcttcgcgga
     5521 cttgtgcatt gatccttcga acccgggttg cctgacctac gaggcatcgg atttcctgat
     5581 cagggatcgc ccggtcagca tccacagtca cttggtcctg ttccaagtcg gatgcgtcgg
     5641 tatcgccgac ttcaacttca ctggattcga caacaacaaa ttcggcgttc tcgtcgaccg
     5701 tctcgagcaa gaatacggcg ccgagcaccc tgtcgtccat tacatcgcag ctatgatgcc
     5761 acaccaagac ccagtcaccg ataaatacac cgtcgcgcag ctccgtgagc ccgagatcgc
     5821 gaagcgtgtt ggcggtgtct cgactttcta catccctccc aaggccagga aagcatcgaa
     5881 cttggacatc ataaggcgcc tagagctctt gcctgctggg caagttcccg acaagaaagc
     5941 gcgtatttac ccggccaacc agtgggagcc cgatgttccc gaagtcgaac cctacagacc
     6001 atctgaccag gctgccatcg ctcagttggc tgaccacgct cctcctgagc aatatcaacc
     6061 tcttgctact tcgaaagcca tgtctgatgt tatgacgaag ttggctttgg atcccaaggc
     6121 actcgccgac tacaaagctg atcaccgcgc ctttgctcaa tctgtccccg acttgacgcc
     6181 tcaggagcgt gcggctttgg agctcggtga ttcgtgggct attcgttgcg cgatgaagaa
     6241 tatgccctcg tcgctcttgg acgctgctcg tgaatccggc gaagaggcat cccaaaacgg
     6301 tttcccatgg gtcatcgtcg ttggtgttat cggtgtcatc ggataagcgg ccgcactcga
     6361 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
     6421 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     6481 gaggggtttt ttgctgaaag gaggaactat atccggat
//
