LOCUS       sequence-175163-        7878 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression of K. stuttgartiensis protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7878)
  AUTHORS   Javidpour P, Deutsch S, Mutalik VK, Hillson NJ, Petzold CJ, Keasling
            JD, Beller HR
  TITLE     Investigation of Proposed Ladderane Biosynthetic Genes from Anammox 
            Bacteria by Heterologous Expression in E. coli.
  JOURNAL   PLoS One. 2016 Mar 14;11(3):e0151087. doi: 
            10.1371/journal.pone.0151087. eCollection 2016.
  PUBMED    26975050
REFERENCE   2  (bases 1 to 7878)
  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: "PLoS One.";
            date: "2016-03-14"; volume: "11(3):e0151087. doi"; pages: " 
            10.1371/journal.pone.0151087. eCollection 2016"
COMMENT     Sequence Label: JPUB_006812
FEATURES             Location/Qualifiers
     source          1..7878
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     563..582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(602..679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     885..904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     988..1007
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        988..1006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1007..1031
                     /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             1046..1068
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1087..1104
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1114..2211
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2185..2208
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2221..2238
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3730..3747
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3800..3818)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3814..3861
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3898..4353
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3985..4004)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4195..4216
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4445..5260)
                     /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(4445..5260)
                     /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     5168..5187
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5252..5271
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5382..5970
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5871..5890
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6124..6141
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6156..6295
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6221..6240)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6381..6403
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6397..6588)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7397..7878,1..601))
                     /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"
ORIGIN
        1 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
       61 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      121 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      181 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      241 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      301 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      361 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
      421 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
      481 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
      541 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
      601 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
      661 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
      721 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
      781 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
      841 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
      901 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
      961 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     1021 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc
     1081 agcagccatc atcatcatca tcacagcagc ggcaaaatcg aagaaggtaa actggtaatc
     1141 tggattaacg gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa
     1201 gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag
     1261 gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc
     1321 tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg
     1381 tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct
     1441 gttgaagcgt tatcgctgat ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg
     1501 gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc
     1561 aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc
     1621 aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa
     1681 gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat
     1741 tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg
     1801 tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc
     1861 ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc
     1921 agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac tgatgaaggt
     1981 ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa
     2041 gagttggcga aagatccacg tattgccgcc actatggaaa acgcccagaa aggtgaaatc
     2101 atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac
     2161 gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac tagcagcggc
     2221 ctggtgccgc gcggcagcca tatgaagaaa ctggttctga tcaacccaca tccaatgggc
     2281 aacgtgggcg aagaaaatgt gagtgttctg aaccaaatgc cggtgaacct gggctacctg
     2341 aaagttctga cgccaggtaa ttggcaagtg gacattatcg atgagaccca agaatacgcc
     2401 attgatgaaa atacgggcga cattaccttt ggtggtgcgg atctggtggg tattaccagc
     2461 gtgagttacc aagccgatcg cgcctacaag attgcgacgg cgtgtcgtaa acgtggtatt
     2521 ccggttatta tgggtggcat ccatgccacg agtaacccag acgaggccgc gaaatacgtt
     2581 gatagcgtgg tgattcgtga aggcatcacc ctgtggagta ccatcattga cgattttgaa
     2641 aagggtcgcc tgcgtaagcg ttatgatggc ggtctgaccc caatggaaat ttactgtaat
     2701 ctgagcccag atcgcaagtt tctgaaagat aaatacaaat accgttatag cggtattatt
     2761 acgaccgcgg gttgtccatt tgcctgtgaa ttttgtagtg tgccacaatt tcagggtcaa
     2821 aaatatcgcg agcgtccagt ggatgatatt ctgaatgaat ttgaaagcat taaaggtcaa
     2881 tatcgtggcc tgattctgac cgacgaaaac ttttacggtc atagcaaaaa gagtaatgaa
     2941 cgtgtgcgta ccctgttcaa aggcatggtg gaacgcggta tctatcagaa ttggtttggt
     3001 ttcaccagtc tgaatatcta caaagatgac gaaaccctgg aatacatggc gaaaagtggt
     3061 tgtgtgggtg tgctgattgg tattgagagc attgaggaag aaagcctgaa gagtatgaac
     3121 aaaaacgtga atctgaaaat tagtgttgaa aactactatg aggccatcgc gaacattcgt
     3181 aagcatggtc tggccgtgtg gggtacgatg gtttttggta atgatgccga caccagcggt
     3241 accttcgaac gcgtggccga ttttatcctg gatagccata ttgatattat gacctgcggc
     3301 atcctgtgtc cgtttatcaa caccccactg taccaccgcc tgaaccgcga gaatcgtctg
     3361 tttcgtacgc atttcccaga agattggaag tattatacca gtcaccacct gacctacgtg
     3421 ctgaaaaagc tgacgctgga tgaaatgatt gaaggtttcc agtacctgta tgatcgtatc
     3481 tttgccaccg aagtgctgcg caaacgcttc catcgtgcca aagacgaact gaataacaac
     3541 atgaatgcgg ccatgtttgc gttccgtgtt aatctggatt ggcagaatgt gtaccaacat
     3601 ctgattcaaa acctgaagga tctgcatacg agtggcgact atgaagcggc cctgaagtgt
     3661 tatgtggata ccgacaaaca aggtaagggt aaaaaactgg cgagtatcga agccgcccca
     3721 taactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     3781 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     3841 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     3901 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3961 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4021 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4081 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     4141 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4201 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     4261 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4321 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     4381 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     4441 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     4501 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     4561 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     4621 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     4681 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     4741 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     4801 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     4861 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     4921 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     4981 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     5041 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     5101 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     5161 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     5221 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     5281 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     5341 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5401 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5461 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5521 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5581 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5641 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5701 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5761 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5821 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5881 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5941 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6001 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     6061 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     6121 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     6181 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     6241 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     6301 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     6361 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     6421 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     6481 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     6541 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     6601 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     6661 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     6721 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     6781 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     6841 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     6901 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     6961 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     7021 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     7081 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     7141 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     7201 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     7261 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     7321 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     7381 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     7441 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     7501 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     7561 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     7621 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     7681 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     7741 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     7801 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     7861 gagtgagata tttatgcc
//
