LOCUS       sequence-175168-        6771 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 6771)
  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 6771)
  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_006820
FEATURES             Location/Qualifiers
     source          1..6771
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     159..178
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     262..281
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        262..280
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    281..305
                     /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             320..342
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             361..378
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             388..405
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1897..1914
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1967..1985)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1981..2028
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2065..2520
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2152..2171)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2362..2383
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2612..3427)
                     /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(2612..3427)
                     /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     3335..3354
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3419..3438
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3549..4137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4038..4057
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4291..4308
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4323..4462
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4388..4407)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4548..4570
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4564..4755)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5564..6646)
                     /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     6608..6627
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6647..6724)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
       61 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
      121 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
      181 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
      241 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
      301 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat gggcagcagc
      361 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatat gaagaaactg
      421 gttctgatca acccacatcc aatgggcaac gtgggcgaag aaaatgtgag tgttctgaac
      481 caaatgccgg tgaacctggg ctacctgaaa gttctgacgc caggtaattg gcaagtggac
      541 attatcgatg agacccaaga atacgccatt gatgaaaata cgggcgacat tacctttggt
      601 ggtgcggatc tggtgggtat taccagcgtg agttaccaag ccgatcgcgc ctacaagatt
      661 gcgacggcgt gtcgtaaacg tggtattccg gttattatgg gtggcatcca tgccacgagt
      721 aacccagacg aggccgcgaa atacgttgat agcgtggtga ttcgtgaagg catcaccctg
      781 tggagtacca tcattgacga ttttgaaaag ggtcgcctgc gtaagcgtta tgatggcggt
      841 ctgaccccaa tggaaattta ctgtaatctg agcccagatc gcaagtttct gaaagataaa
      901 tacaaatacc gttatagcgg tattattacg accgcgggtt gtccatttgc ctgtgaattt
      961 tgtagtgtgc cacaatttca gggtcaaaaa tatcgcgagc gtccagtgga tgatattctg
     1021 aatgaatttg aaagcattaa aggtcaatat cgtggcctga ttctgaccga cgaaaacttt
     1081 tacggtcata gcaaaaagag taatgaacgt gtgcgtaccc tgttcaaagg catggtggaa
     1141 cgcggtatct atcagaattg gtttggtttc accagtctga atatctacaa agatgacgaa
     1201 accctggaat acatggcgaa aagtggttgt gtgggtgtgc tgattggtat tgagagcatt
     1261 gaggaagaaa gcctgaagag tatgaacaaa aacgtgaatc tgaaaattag tgttgaaaac
     1321 tactatgagg ccatcgcgaa cattcgtaag catggtctgg ccgtgtgggg tacgatggtt
     1381 tttggtaatg atgccgacac cagcggtacc ttcgaacgcg tggccgattt tatcctggat
     1441 agccatattg atattatgac ctgcggcatc ctgtgtccgt ttatcaacac cccactgtac
     1501 caccgcctga accgcgagaa tcgtctgttt cgtacgcatt tcccagaaga ttggaagtat
     1561 tataccagtc accacctgac ctacgtgctg aaaaagctga cgctggatga aatgattgaa
     1621 ggtttccagt acctgtatga tcgtatcttt gccaccgaag tgctgcgcaa acgcttccat
     1681 cgtgccaaag acgaactgaa taacaacatg aatgcggcca tgtttgcgtt ccgtgttaat
     1741 ctggattggc agaatgtgta ccaacatctg attcaaaacc tgaaggatct gcatacgagt
     1801 ggcgactatg aagcggccct gaagtgttat gtggataccg acaaacaagg taagggtaaa
     1861 aaactggcga gtatcgaagc cgccccataa ctcgagcacc accaccacca ccactgagat
     1921 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     1981 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2041 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2101 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2161 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2221 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2281 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2341 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2401 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2461 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2521 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2581 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     2641 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     2701 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     2761 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     2821 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     2881 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     2941 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     3001 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3061 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     3121 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3181 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     3241 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3301 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3361 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     3421 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     3481 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3541 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3601 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3661 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3721 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3781 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3841 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3901 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3961 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4021 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4081 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4141 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4201 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4261 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4321 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4381 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4441 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4501 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4561 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4621 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4681 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4741 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     4801 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     4861 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4921 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     4981 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     5041 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     5101 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     5161 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     5221 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     5281 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     5341 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     5401 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     5461 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5521 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5581 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5641 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     5701 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     5761 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     5821 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     5881 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     5941 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     6001 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     6061 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     6121 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     6181 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     6241 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     6301 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     6361 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     6421 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     6481 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     6541 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     6601 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     6661 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     6721 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca g
//
