LOCUS       sequence-177975-        7914 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for expression of N-8xHis-StrepII-MBP-3607.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7914)
  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 7914)
  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: Ppj217(JBEI-13454)
FEATURES             Location/Qualifiers
     source          1..7914
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(270..1352)
                     /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     1314..1333
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1353..1430)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1636..1655
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1739..1758
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1739..1757
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1758..1782
                     /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             1797..1819
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1829..1852
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1862..1885
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1895..2992
                     /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     2966..2989
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3011..3031
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4517..4534
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4587..4605)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4601..4648
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4685..5140
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4772..4791)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4982..5003
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5232..6047)
                     /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(5232..6047)
                     /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     5955..5974
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6039..6058
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6169..6757
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6658..6677
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6911..6928
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6943..7082
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7008..7027)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7168..7190
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7184..7375)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
       61 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
      121 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
      181 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
      241 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
      301 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
      361 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
      421 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
      481 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
      541 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
      601 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
      661 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
      721 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
      781 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
      841 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
      901 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
      961 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     1021 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     1081 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     1141 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     1201 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     1261 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     1321 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     1381 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     1441 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     1501 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     1561 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     1621 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     1681 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
     1741 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     1801 tttaacttta agaaggagat ataccatgca tcaccaccac catcaccacc atggttctgg
     1861 atggagccat ccgcagtttg aaaaaggatc aggtaaaatc gaagaaggta aactggtaat
     1921 ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga aattcgagaa
     1981 agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca
     2041 ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg
     2101 ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc aggacaagct
     2161 gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc
     2221 tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctg
     2281 ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt
     2341 caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt
     2401 caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa
     2461 agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga
     2521 ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc
     2581 gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgac
     2641 cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc
     2701 cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga ctgatgaagg
     2761 tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga
     2821 agagttggcg aaagatccac gtattgccgc cactatggaa aacgcccaga aaggtgaaat
     2881 catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa
     2941 cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga ctgggtctgg
     3001 atcaggctct gaaaaccttt acttccaggg cggatccggt atgagtagca ccaaaccaat
     3061 taaaccacat attcaaagta ccaagattga cacgctgtat ccgagttacg atgtgattgt
     3121 tattggtgcc ggtatcgccg gtctgatttg tgccgccttc ctggccaaaa gtggtaagaa
     3181 agccctgctg attgagcaac atttcattcc aggtggctac tgtaccagct ttaaacgtaa
     3241 gggtttcctg tttgatgcgg cggttcatca tattggcggt tgcggtaaat ggagcgtggt
     3301 gggtcgttgt ttcaaaatgc tgggcatcga aatggacttt taccatctgg acccaatgga
     3361 taatctgatc ttcccaacgt ttagcattga aattccagcc gatctggatg aatacctgct
     3421 gcgcctgcaa aagcgttacc cacatgaaaa ggataacatt aagtggtttt ttcaagactt
     3481 tatccgcctg taccgcgcca ccttccataa tgagaaaagt caaatcatcg agaaatacaa
     3541 agatatgacg tacggcatga tgctggatca tttctttgat gatagtgaac tgaaaatggt
     3601 tctgagcggc cagtggggtt acgtgggtct ggccccgagc aaggccagcg ccattgccat
     3661 gagccaaatg atgattaact atctgaaaga tggtgccttt tttccaaagg gtggtaccca
     3721 agaatttgcc aatgccattt ttcgtaagtt tatcgatttc ggcggtcatg ttatgctgag
     3781 tagtaaagtg aagaaaatcc tgaagaaggg cccaagtgtt aatggtgtga aactgaatga
     3841 cgataaggaa attcatagca gtgttgtggt gagcaatatc gatgcgcgtc agacctttta
     3901 tcatctgctg aacgacggta atgatgtgga tgccgtttac ctgaaaaaca ttgagggtat
     3961 gaaagaaagt tgtagttttt ttctgctgta cctgggtatc ggtaaaggca ttgacctgaa
     4021 tacgctgaag cgcggcttct acctgagtgg tagcaatttc gacagcatcg acagtgaatg
     4081 gatgtatatt agtgtgccaa ccagtatttg tcaagcgctg agtccacaaa ataagcaaat
     4141 tatcagtatc gtggtgtaca agaaagaaga gttctacaaa aatgtgaccg attgggaact
     4201 gctgaaacag aatattgtga gcagtacgct ggagcgcctg gaaagttacc tgccggatat
     4261 taagaaacat attgagatca ttgaagttgc caccccaaaa accctggaac gctacaccct
     4321 gaacacgaac ggtgccgcgt atggttgggc cgtgagtgtt gaccaaatct gggaacagcg
     4381 tctgagccca acgacgccga ttagtggtct gtatctggcg ggtcattgga cgcgcccagg
     4441 tccaggtatc tgcgccgtgg ttagtagtgg ttggagtgtg gccaacctga ttctgaaaaa
     4501 ttggcgttaa ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     4561 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     4621 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     4681 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4741 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4801 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4861 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     4921 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4981 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     5041 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     5101 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     5161 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5221 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     5281 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     5341 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     5401 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     5461 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     5521 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     5581 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     5641 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     5701 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     5761 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     5821 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     5881 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     5941 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     6001 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     6061 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     6121 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     6181 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     6241 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     6301 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     6361 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     6421 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     6481 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     6541 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     6601 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     6661 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     6721 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     6781 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     6841 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     6901 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     6961 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     7021 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     7081 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     7141 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     7201 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     7261 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     7321 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     7381 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     7441 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     7501 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     7561 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     7621 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     7681 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     7741 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     7801 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     7861 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gctt
//
