LOCUS       sequence-175166-        6745 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 6745)
  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 6745)
  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_006826
FEATURES             Location/Qualifiers
     source          1..6745
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     969..988
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1008..1085)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1291..1310
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1394..1413
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1394..1412
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1413..1437
                     /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             1452..1474
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1493..1510
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1520..1537
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3003..3020
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3073..3091)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3087..3134
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3171..3626
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3258..3277)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3468..3489
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3718..4533)
                     /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(3718..4533)
                     /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     4441..4460
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4525..4544
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4655..5243
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5144..5163
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5397..5414
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5429..5568
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5494..5513)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5654..5676
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5670..5861)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6670..6745,1..1007))
                     /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 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
       61 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
      121 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
      181 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
      241 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
      301 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
      361 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
      421 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
      481 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      541 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      601 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      661 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      721 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      781 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      841 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      901 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      961 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     1021 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     1081 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     1141 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     1201 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     1261 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     1321 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     1381 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1441 ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca
     1501 tcatcatcac agcagcggcc tggtgccgcg cggcagccat atgaagaaga tgatgctgat
     1561 taacccacat aaaccaggtc gtcatggtga ggaaagcatt acggtgattg tgcaaatgcc
     1621 gctgaatctg gcctacatta aagccctgac cccaggcgat tgggaatttg atgtgattga
     1681 cgaaaacatc gaactggcca ttgatgataa cggtgaactg accttcgcgc cagtggatct
     1741 ggtgtgtatt accagtgtga cctaccaaag tccacgtgcc tacaagattg ccaccgcctg
     1801 taaacgtaaa ggtatgaccg tgatcatggg tggtattcat gcgagcgtga tgccagaaga
     1861 agccagtaag tacgttgaca ccgtgtttat tggtgaagcc gaggaagtgt ggccgcgtgt
     1921 tattaaggat ttcgaagccg gtaaactgaa aaaagtttat gacggtggtc tgccaccact
     1981 gaacctgatg aaacgtgtgt ttccagatcg tgaatttctg cgcaagaaat acaattacaa
     2041 atttagtagt attgtgacca ccaagggttg tccaaattac tgtgatttct gtagcgtgcc
     2101 aacctttcaa ggccgcaaat ttcgtgaacg tccatacgaa gatgtgctgg aagagctggc
     2161 ggcgaccgac tacaagggcc tgatgctggc cgaagataac ttctatggtc atggcaaacg
     2221 tagtaacgaa cgtgcccgta acctgtttaa aggcatggtg gaacgtaatc tgcaaaaaga
     2281 ctggctgggt tttaccgccc tgaacattag tcaagacaaa gaaaccctgg actacatggc
     2341 caaaagtggt tgttttggta tgctgatggg tatcgagagt accaatgaaa ccgttctgga
     2401 gaaaatgaac aaacaagtta atctgaaact gggtaccgaa agctattatg actgtatcca
     2461 aaagatccat gatgccggtc tggttacctg gggcagtgtg gtgtttggtg cggatggtga
     2521 tggtaaagat agtttcaagc gcatgaccga ttttatcctg gaaaataaca ttgatattct
     2581 gacgtttggc attaattgtc catttccaaa aacgcaactg tacaaacgcc tggatagtga
     2641 aaaacgtatt tttcgtaaga attacccaga tgactgggaa tattacgaca cggcccatgt
     2701 ggtgcatcgt ctggtggata tgacgctgga ggactttatt gagggtatgc aatacgtgta
     2761 cgaccatatt tacgccggtg acaatctgcg taagcgtttt cgcaatagta ttaagacgac
     2821 gaataatcca cgtaacagta tgtttgcctt tcgtgttggc agtgattggc aacaagtgtt
     2881 tgaccaagtt ctggagaacc tgcgcctgct gtatgatagt ggtgattact ataaagatta
     2941 ttacaagagt aatagcgtga gtgtgagtaa aaaaccgctg gttgaaagta tcacgacgta
     3001 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     3061 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     3121 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     3181 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3241 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3301 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3361 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3421 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3481 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3541 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     3601 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     3661 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     3721 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     3781 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     3841 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     3901 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     3961 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     4021 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     4081 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     4141 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     4201 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     4261 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     4321 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     4381 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     4441 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     4501 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     4561 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4621 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4681 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4741 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4801 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4861 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4921 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4981 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5041 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5101 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5161 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5221 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5281 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5341 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5401 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     5461 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5521 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     5581 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5641 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5701 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     5761 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     5821 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     5881 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     5941 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     6001 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     6061 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     6121 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     6181 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     6241 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     6301 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     6361 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     6421 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     6481 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     6541 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     6601 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     6661 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6721 atcggccaac gcgcggggag aggcg
//
