LOCUS       sequence-175167-        7857 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 7857)
  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 7857)
  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_006818
FEATURES             Location/Qualifiers
     source          1..7857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     760..779
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(799..876)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1082..1101
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1185..1204
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1185..1203
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1204..1228
                     /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             1243..1265
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1284..1301
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1311..2408
                     /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     2382..2405
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2418..2435
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3906..3923
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3976..3994)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3990..4037
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4074..4529
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4161..4180)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4371..4392
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4621..5436)
                     /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(4621..5436)
                     /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     5344..5363
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5428..5447
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5558..6146
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6047..6066
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6300..6317
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6332..6471
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6397..6416)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6557..6579
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6573..6764)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7573..7857,1..798))
                     /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 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
       61 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
      121 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
      181 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
      241 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
      301 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
      361 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
      421 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
      481 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
      541 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
      601 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
      661 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
      721 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
      781 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
      841 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
      901 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
      961 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     1021 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     1081 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     1141 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     1201 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     1261 ggagatatac catgggcagc agccatcatc atcatcatca cagcagcggc aaaatcgaag
     1321 aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc gctgaagtcg
     1381 gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg gataaactgg
     1441 aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac
     1501 acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag
     1561 cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga
     1621 ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga
     1681 acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta
     1741 agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg attgctgctg
     1801 acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg
     1861 ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa aacaaacaca
     1921 tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc gaaacagcga
     1981 tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg aattatggtg
     2041 taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc gtgctgagcg
     2101 caggtattaa cgccgccagt ccgaacaaag agctggcaaa agagttcctc gaaaactatc
     2161 tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt gccgtagcgc
     2221 tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccact atggaaaacg
     2281 cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc
     2341 gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg
     2401 cgcagactag cagcggcctg gtgccgcgcg gcagccatat gaagaagatg atgctgatta
     2461 acccacataa accaggtcgt catggtgagg aaagcattac ggtgattgtg caaatgccgc
     2521 tgaatctggc ctacattaaa gccctgaccc caggcgattg ggaatttgat gtgattgacg
     2581 aaaacatcga actggccatt gatgataacg gtgaactgac cttcgcgcca gtggatctgg
     2641 tgtgtattac cagtgtgacc taccaaagtc cacgtgccta caagattgcc accgcctgta
     2701 aacgtaaagg tatgaccgtg atcatgggtg gtattcatgc gagcgtgatg ccagaagaag
     2761 ccagtaagta cgttgacacc gtgtttattg gtgaagccga ggaagtgtgg ccgcgtgtta
     2821 ttaaggattt cgaagccggt aaactgaaaa aagtttatga cggtggtctg ccaccactga
     2881 acctgatgaa acgtgtgttt ccagatcgtg aatttctgcg caagaaatac aattacaaat
     2941 ttagtagtat tgtgaccacc aagggttgtc caaattactg tgatttctgt agcgtgccaa
     3001 cctttcaagg ccgcaaattt cgtgaacgtc catacgaaga tgtgctggaa gagctggcgg
     3061 cgaccgacta caagggcctg atgctggccg aagataactt ctatggtcat ggcaaacgta
     3121 gtaacgaacg tgcccgtaac ctgtttaaag gcatggtgga acgtaatctg caaaaagact
     3181 ggctgggttt taccgccctg aacattagtc aagacaaaga aaccctggac tacatggcca
     3241 aaagtggttg ttttggtatg ctgatgggta tcgagagtac caatgaaacc gttctggaga
     3301 aaatgaacaa acaagttaat ctgaaactgg gtaccgaaag ctattatgac tgtatccaaa
     3361 agatccatga tgccggtctg gttacctggg gcagtgtggt gtttggtgcg gatggtgatg
     3421 gtaaagatag tttcaagcgc atgaccgatt ttatcctgga aaataacatt gatattctga
     3481 cgtttggcat taattgtcca tttccaaaaa cgcaactgta caaacgcctg gatagtgaaa
     3541 aacgtatttt tcgtaagaat tacccagatg actgggaata ttacgacacg gcccatgtgg
     3601 tgcatcgtct ggtggatatg acgctggagg actttattga gggtatgcaa tacgtgtacg
     3661 accatattta cgccggtgac aatctgcgta agcgttttcg caatagtatt aagacgacga
     3721 ataatccacg taacagtatg tttgcctttc gtgttggcag tgattggcaa caagtgtttg
     3781 accaagttct ggagaacctg cgcctgctgt atgatagtgg tgattactat aaagattatt
     3841 acaagagtaa tagcgtgagt gtgagtaaaa aaccgctggt tgaaagtatc acgacgtaac
     3901 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
     3961 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     4021 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     4081 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4141 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4201 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4261 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     4321 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4381 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     4441 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4501 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     4561 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     4621 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     4681 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     4741 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     4801 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     4861 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     4921 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     4981 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     5041 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     5101 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     5161 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     5221 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     5281 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     5341 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     5401 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     5461 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     5521 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5581 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     5641 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     5701 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     5761 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     5821 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5881 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     5941 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6001 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6061 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6121 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6181 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     6241 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     6301 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     6361 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     6421 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     6481 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     6541 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     6601 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     6661 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     6721 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     6781 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     6841 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     6901 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     6961 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     7021 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     7081 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     7141 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     7201 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     7261 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     7321 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     7381 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     7441 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     7501 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     7561 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     7621 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     7681 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     7741 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     7801 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatc
//
