LOCUS       sequence-273457-        6759 bp    DNA     circular SYN 15-APR-2025
DEFINITION  expresses KinO1 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6759)
  AUTHORS   Wang P, Hong GJ, Wilson MR, Balskus EP
  TITLE     Production of Stealthin C Involves an S-N-Type Smiles Rearrangement.
  JOURNAL   J Am Chem Soc. 2017 Mar 1;139(8):2864-2867. doi: 
            10.1021/jacs.6b10586. Epub 2017 Feb 16.
  PUBMED    28191843
REFERENCE   2  (bases 1 to 6759)
  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"; doi: 
            "10.1021/jacs.6b10586"; journalName: "J Am Chem Soc"; date: 
            "2017-03-1- 1"; volume: "139"; issue: "8"; pages: "2864-2867"
COMMENT     Sequence Label: pET28a-KinO1
FEATURES             Location/Qualifiers
     source          1..6759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    39..63
                     /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             78..100
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             119..136
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             146..163
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1643..1660
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1713..1731)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1727..1774
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1811..2266
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1898..1917)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2108..2129
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2358..3173)
                     /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(2358..3173)
                     /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     3081..3100
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3165..3184
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3295..3883
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3784..3803
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4037..4054
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4069..4208
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4134..4153)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4294..4316
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4310..4501)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5310..6392)
                     /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     6354..6373
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6393..6470)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6676..6695
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
       61 tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca
      121 tcatcatcat catcacagca gcggcctggt gccgcgcggc agccatatgg ataactttga
      181 cgcggacgta attatcgcgg gagccggccc cactggactc atgctcgcag gcgaactgcg
      241 cctgaacggc gtgtccgtga ttgtcgtcga tcgccttgcc gagccgatac agcagtcgcg
      301 tgcgctggga ttttcggccc gtaccatcga ggaattcggc cagcgcggac tgctgtcccg
      361 tttcggtgaa gtcgacgtca ttccggtcgg tcatttcggc ggagtgtcca tcgattaccg
      421 tctggtcgag ggcggttcgt acggagcgcg cggcattccc cagtcgcgca ccgagggcgt
      481 gctggcgggc tgggccggac agctcggcgc cgaggtgcgg cgcggggtcg aggtcacggg
      541 cctggacaac ggcgccgacg gcgtgagcgt ggaggtcagc accgccgagg ggcccgccac
      601 gctgcgcggc cgctacctgg tgggcgcgga cggcgcccgc agtgcggtgc gcaagctcgc
      661 gggcatcgac ttcccgggca ccgacccggc catcgagctc cggttcgccg acatcagcgg
      721 ggtgccgttg cgcccgcgct tcagcggcga gcgggtgccc ggcggcatgg tcatggtgct
      781 gccgctcggc ccggagcgct gccggatcgt ctacttcgac cgcagcgagc cgttgcgcaa
      841 gagcccggac ccgatcacct tcgacgaggt ggccgaggcg ttcaagcggc tgtccggcga
      901 ggacatcagc ggcgccaccg tgcactgggt ctccaccacc accgatgtga gccggcaggc
      961 cgccgagtac cgcaggggcc gggtcttcct ggccggcgac gccgcccaca tccatctgcc
     1021 catcggcgcc cagggaatga gcgccggcgt gcaggacgcg gtcaacctcg gctggaagct
     1081 cgccctggag atcaagggcc aggctcccga gggcctgctc gacacgtacc actccgagcg
     1141 gcacccggtc ggcgcgcggg tcctcaccaa cacgctcgcc cagcggatcc tctacctcgg
     1201 cggcgacgag atcaccccgc tgctcgatgt gttcaccgag ctcaccgggt tcgaggacgt
     1261 ccagaagacg ctgatcggca tggtcaccgg cctggacatc cgccatgacg tgggcgaggg
     1321 cgaccatccg ctcctcggcc gccgtctcaa ggacgaggag ctggtggtcg acggcaagaa
     1381 gaccaccaac ttcgaactcc tcgcggccgg caaggccgtt ctgttcaacc tcaccgatga
     1441 cccccacctg cgggagctcg ccgcgggctg ggccgaccgt gtcaccacgg tcaccgccga
     1501 gcagcacgac tgcgacaacg gtctggacgc cttcctggtc cgtcccgacg gctatgtcgc
     1561 ctgggtcgcc ccttccgcgt cgcgcacgga ggggctcgcc gaagcactca accgatggtt
     1621 cggccggccc aactgactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
     1681 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     1741 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
     1801 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     1861 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     1921 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     1981 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2041 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2101 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     2161 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     2221 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     2281 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2341 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     2401 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     2461 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     2521 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     2581 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     2641 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     2701 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     2761 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     2821 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     2881 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     2941 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     3001 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     3061 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     3121 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     3181 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     3241 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3301 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3361 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3421 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     3481 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3541 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3601 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3661 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3721 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3781 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3841 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3901 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3961 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4021 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     4081 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     4141 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     4201 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4261 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4321 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     4381 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     4441 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     4501 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     4561 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     4621 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     4681 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     4741 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     4801 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     4861 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     4921 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     4981 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     5041 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     5101 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     5161 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     5221 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     5281 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5341 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5401 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     5461 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     5521 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     5581 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     5641 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     5701 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     5761 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5821 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5881 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5941 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     6001 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     6061 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     6121 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     6181 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     6241 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     6301 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     6361 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     6421 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     6481 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     6541 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     6601 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     6661 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     6721 ggtgatgccg gccacgatgc gtccggcgta gaggatcga
//
