LOCUS       sequence-344760-        9182 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mevalonate production pathway with J306 scRNA on pBBR1-GmR plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9182)
  AUTHORS   Kiattisewee C, Dong C, Fontana J, Sugianto W, Peralta-Yahya P, 
            Carothers JM, Zalatan JG
  TITLE     Portable bacterial CRISPR transcriptional activation enables 
            metabolic engineering in Pseudomonas putida.
  JOURNAL   Metab Eng. 2021 Apr 27. pii: S1096-7176(21)00063-X. doi: 
            10.1016/j.ymben.2021.04.002.
  PUBMED    33930546
REFERENCE   2  (bases 1 to 9182)
  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: "Metab Eng. 
            2021 Apr 27. pii: S1096-7176(21)00063-X. doi: 
            10.1016/j.ymben.2021.04.002."
COMMENT     Sequence Label: pPPC030.306
FEATURES             Location/Qualifiers
     source          1..9182
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1617..2150)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(2339..2344,2345..2361,2362..2367))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     terminator      complement(2533..2560)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2576..2647)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             6276..6287
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(6297..6331)
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     protein_bind    6624..6645
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6660..6665,6666..6683,6684..6690)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6698..6714
                     /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)."
     primer_bind     6703..6725
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6722..6738
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6722..6738
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6757..6777
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6759..6777
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     terminator      complement(6822..6868)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     7021..7038
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(7094..7111)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(7127..7156)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_RNA        complement(7179..7197)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        complement(7281..7315)
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     primer_bind     complement(7329..7348)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7330..7348)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7358..7375)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7358..7374)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7367..7389)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(8083..8745)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      join(8746..9182,1..335)
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
ORIGIN
        1 tgcccccact cgcataggct tgggtcgttc caggcgcgtc aaggccaagc cgctgcgcgg
       61 tcgctgcgcg agccttgacc cgccttccac ttggtgtcca accggcaagc gaagcgcgca
      121 ggccgcaggc cggaggcttt tccccagaga aaattaaaaa aattgatggg gcaaggccgc
      181 aggccgcgca gttggagccg gtgggtatgt ggtcgaaggc tgggtagccg gtgggcaatc
      241 cctgtggtca agctcgtggg caggcgcagc ctgtccatca gcttgtccag cagggttgtc
      301 cacgggccga gcgaagcgag ccagccggtg gccgctcgcg gccatcgtcc acatatccac
      361 gggctggcaa gggagcgcag cgaccgcgca gggcgaagcc cggagagcaa gcccgtaggg
      421 cgccgcagcc gccgtaggcg gtcacgactt tgcgaagcaa agtctagtga gtatactcaa
      481 gcattgagtg gcccgccgga ggcaccgcct tgcgctgccc ccgtcgagcc ggttggacac
      541 caaaagggag gggcaggcat ggcggcatac gcgatcatgc gatgcaagaa gctggcgaaa
      601 atgggcaacg tggcggccag tctcaagcac gcctaccgcg agcgcgagac gcccaacgct
      661 gacgccagca ggacgccaga gaacgagcac tgggcggcca gcagcaccga tgaagcgatg
      721 ggccgactgc gcgagttgct gccagagaag cggcgcaagg acgctgtgtt ggcggtcgag
      781 tacgtcatga cggccagccc ggaatggtgg aagtcggcca gccaagaaca gcaggcggcg
      841 ttcttcgaga aggcgcacaa gtggctggcg gacaagtacg gggcggatcg catcgtgacg
      901 gccagcatcc accgtgacga aaccagcccg cacatgaccg cgttcgtggt gccgctgacg
      961 caggacggca ggctgtcggc caaggagttc atcggcaaca aagcgcagat gacccgcgac
     1021 cagaccacgt ttgcggccgc tgtggccgat ctagggctgc aacggggcat cgagggcagc
     1081 aaggcacgtc acacgcgcat tcaggcgttc tacgaggccc tggagcggcc accagtgggc
     1141 cacgtcacca tcagcccgca agcggtcgag ccacgcgcct atgcaccgca gggattggcc
     1201 gaaaagctgg gaatctcaaa gcgcgttgag acgccggaag ccgtggccga ccggctgaca
     1261 aaagcggttc ggcaggggta tgagcctgcc ctacaggccg ccgcaggagc gcgtgagatg
     1321 cgcaagaagg ccgatcaagc ccaagagacg gcccgagacc ttcgggagcg cctgaagccc
     1381 gttctggacg ccctggggcc gttgaatcgg gatatgcagg ccaaggccgc cgcgatcatc
     1441 aaggccgtgg gcgaaaagct gctgacggaa cagcgggaag tccagcgcca gaaacaggcc
     1501 cagcgccagc aggaacgcgg gcgcgcacat ttccccgaaa agtgccacct gcggcgttgt
     1561 gacaatttac cgaacaactc cgcggccggg aagccgatct cggcttgaac gaattgttag
     1621 gtggcggtac ttgggtcgat atcaaagtgc atcacttctt cccgtatgcc caactttgta
     1681 tagagagcca ctgcgggatc gtcaccgtaa tctgcttgca cgtagatcac ataagcacca
     1741 agcgcgttgg cctcatgctt gaggagattg atgagcgcgg tggcaatgcc ctgcctccgg
     1801 tgctctccgg agactgcgag atcatagata tagatctcac tacgcggctg ctcaaacttg
     1861 ggcagaacgt aagccgcgag agcgccaaca accgcttctt ggtcgaaggc agcaagcgcg
     1921 atgaatgtct tactacggag caagttcccg aggtaatcgg agtccggctg atgttgggag
     1981 taggtggcta cgtctccgaa ctcacgaccg aaaagatcaa gagcagcccg catggatttg
     2041 acttggtcag ggccgagcct acatgtgcga atgatgccca tacttgagcc acctaacttt
     2101 gttttagggc gactgccctg ctgcgtaaca tcgttgctgc tgcgtaacat cgttgctgct
     2161 ccataacatc aaacatcgac ccacggcgta acgcgcttgc tgcttggatg cccgaggcat
     2221 agactgtaca aaaaaacagt cataacaagc catgaaaacc gccactgcgc cgttaccacc
     2281 gctgcgttcg gtcaaggttc tggaccagtt gcgtgagcgc atacgctact tgcattacag
     2341 tttacgaacc gaacaggctt atgtcaactg ggttcgtgcc ttcatccgtt tccacggtgt
     2401 gcgtccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag
     2461 gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag
     2521 tttttatata aacgcagaaa ggcccacccg aaggtgagcc agtgtgactc tagtagagag
     2581 cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttcgactga gcctttcgtt
     2641 ttatttgatg cctggagatc cttactcgag tttttaatta cgatagctac gcacggtgtt
     2701 attaatggcg ctaatgctat attttaattc gtcttccagg gtctgatcaa tatcggtatc
     2761 cagggtttct gcaaacattg cttcatattc tgcaatgctc agttcggtac gattatccag
     2821 cagggccaga tgggtttctt tctgcagatg attctgataa cctgcaacca gttcaccggt
     2881 aaagaattct gcaacggcac cgctaccata gctaaacaga ccaatctgat taccagcggt
     2941 cagggtggtt gcattttcca gcaggctaat cagacccaga tacaggctac cggtatacag
     3001 attacccaca cgacggctat agataatgct ttcttcataa cgtgccagga tacgttcttg
     3061 ttcggcttcg gtctgatcgc taattttcgc cagcagtgct tttttaccca ttttggtata
     3121 cggaatatga aaggccagtg catcataatc tgcgaaatcc agaccggtac gttttttatg
     3181 ttcatcccac acctgtgcaa agctctgaat ataggtttca ttgctcagcg gaccatcaac
     3241 catcggatac ggatgaccgg tcggacgcca aaaatcatag atatcctgcg tcagcataac
     3301 attatcctct ttcagtgcca gaatacgcgg ttcgcttgca accagcattg caactgcacc
     3361 ggcaccctgg gtcggttcac caccgctatt cagaccatat ttggcgatat ctgctgcaac
     3421 aaccagaact tttttatccg gatgcagtgc aacatgattt tttgccagct gcagacctgc
     3481 ggttgcgccg taacaacctt ctttaatttc aaagctacgt gcaaacggct gaatacccat
     3541 cagacgatgc agaacaactg cggctgcttt gctttcatca atgctgcttt cggtgccaac
     3601 aataaccata tcgatggctt ctttatcttc tttggtcaga attgcttctg ctgcatttgc
     3661 tgcaaaggta acaatatcct ggctaatcgg attaacggcc atctgatcct gaccaatacc
     3721 aatatgaaat ttacccggat caacattacg tgcttcggcc agtgcggtca tgtcgatata
     3781 gtaaggcgga acaaaaaagc tgattttgtc gatgccaatg gtcatttttt gcactcctca
     3841 tattttgtcg ctaatcatta ctgtttacgc agatcattca gaattgccag ggcacgatcc
     3901 tgattcattg ttttctggcg tttcagttgc tgtgcaacgg cttcaacttc tttaccggtc
     3961 gcacccacgg tcatggccag tgaacgtgcc tgcagtgcca tgtgaccttt ctgaatgcct
     4021 tcgctaacca gtgcacgcag tgctgccaga ttctgtgcca gaccaactgc tgcaacaaca
     4081 cggctcagtt cttttgcatc ggtaactgcc agcagatcgg ctgctgcctg gcttttcggc
     4141 agaactttgg tggcaccacc aacggttgcc agtgccagcg gaacgctaat ttcaccaatc
     4201 agctgctcac catccagggt ccagctggtc agaccctgat aacgaccttc tttaactgca
     4261 aatgcatgac agcttgcgct aactgcacgg gtatcattac cggtggccag cacaactgct
     4321 tcaatgccat tcataatacc tttattatgg gtaacggcac gatacggatc caggctggca
     4381 taacggcttg ccagaacaat tttttcggca atttcacgac cattactacc tttgctcaga
     4441 cggctaaccg gaattgctgt tttcatggta acaacgcttt cggtggcata gttactcagg
     4501 atgctaaaca ggattttttg ttctgcaaac cattcacgaa acagttcggc aacaccttcc
     4561 agcattgcat taacaatatt ggcacccatt gcgtctttaa catccaccag aaaatccacg
     4621 ctaacaaaac tttcatcaaa tgcacgatac tgcagatcac gcagaccacc accgcgttta
     4681 acaatgctcg gataactcag ttctgcctgc tggaaaattt ctgtttcacg aacctgcagt
     4741 tcatcaatca ggctttctgc atcggcaaca tcataaaaaa caatctgacc acgcatcaga
     4801 cgctgctgat tcacggtttt aaagccctgt gcaatttttg caccattgct cagggctgca
     4861 ataacgctcg gttcttcggt tgccatcgga accagataat ccgtttcatc cacggtcaga
     4921 tgcagaccaa cacccatcgg cacttcggtt tcgctgatct gattttcaat catgtggttg
     4981 gcaatctggc tgctcagtgc ggtgttttca aattcttttt tggtatctgc gctaatctga
     5041 ccttcattca gcaggctggc cagacgttct tccggactca tctgataaaa acggctgttt
     5101 tttttctgtt gagggcgttc cagcagcatt gccagaccca ggccaccacc aatacacagg
     5161 cttgcaacgc cgtatttttt ctctttctga ttcagctgat agctcaggct ggtcagcaga
     5221 cgtgcaccgg ttgcaccaat tgcatgaccc aggctgatgc caccgccata aatgttaact
     5281 ttttcttccg gcagtgccag ttcacgctga acaacaatgc tggttgcggc aaatgcttca
     5341 ttaatttcgt acaggtcgat ttcttcggtg gtcagctgat tacgtgccag cagtttctga
     5401 attgctttaa tcgggctaat acccatatat gccggatcaa taccaacttc aacgctatca
     5461 cgaataattg ccagatacgg cagaccatgt gcttcggcat attcttggct ggcaataatc
     5521 agtgcgcttg caccatcatt aatggtactt gcattgcctg cggtaacggt gccatcttct
     5581 ttaaacaccg ttttcagtgt acccagtttt tcaacgctgc tattcggacg aataccttca
     5641 tctttttcaa ccagggtgcc gctaacttcc agcggtgcaa tttcatctgc gaaaatacct
     5701 tcggcctgtg cctgtgctgc tttcagctgg ctatgaacgc taaactgatc ctgttcttca
     5761 cgggtcacat gatatttttc tgcaacattt tctgctgtca gacccattgc ctgaccgcta
     5821 aatgcatcgg tcagaccatc atacatcatg ctgctaaacg gtgcatcata gctttcggtt
     5881 tcataattga aacgctgcag tttcggtgcc tggctcatat tttcaatacc accggcaatc
     5941 agaacttcgg cttcgcccag ctggatcagc tgttttgcca gaataactgc tttcataccg
     6001 ctaccacaaa cttcattaac ggtcattgcc ggaatttcat ggctcagacc gctattaatt
     6061 gcaatctgac gtgccggatt ctgaccatta cctgcctgca gaacattgcc aaaaatcacc
     6121 tgatcaattt cttcgctaat ggtgctatga cgtttcagca gctgggtggt aacatgggtg
     6181 cccagatcaa ctgcgctaac ctggctcagg ctacctttgt atttaccaat cggggtacgc
     6241 agtgcatcaa taatcaccac ggttttcatg gtacctttct cctctttaat gaattcgcta
     6301 gcacaatccc taggactgag ctagctgtca atctataatc gcaacttcaa gacgacgttg
     6361 tgtccagaac gctccgtagg agaagtgagg agacgagcga acgcgtcgta cgagctttat
     6421 gcatctttca cagctcgtag cgatgattat gacatcgcag tgagcaactg aataagcgct
     6481 gcgtgaatga tcgcaaatgc tgacgtcgga attgccatgc gcccaatacg caaaccgcct
     6541 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     6601 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     6661 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     6721 acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa
     6781 caaaagctgg tctagcttaa gagttcaccg acaaacaaca gataaaacga aaggcccagt
     6841 ctttcgactg agcctttcgt tttatttgat gcctggcagt tccctactct cgcatgggga
     6901 gaccccacac taccatcggc gctacggcgt ttcacttctg agttcggcat ggggtcaggt
     6961 ggggccaccg cgctactgcc gccaggcaaa ttctgtttta tcagaccgct tctgcgttct
     7021 gatttaatct gtatcaggct gaaaatcttc tctcatccgc caaaacagcc aagctggaga
     7081 ccgtttaaac tcaatgatga tgatgatgat ggtcgacggc gctattcaga tcctcttctg
     7141 agatgagttt ttgttcgggc ccaagcttca aaaaaagcac atgggtgatc ctcatgtgcc
     7201 actttttcaa gttgataacg gactagcctt attttaactt gctatttcta gctctaaaac
     7261 acggagcgtt ctggacacaa actagtatta tacctaggac tgagctagct gtcaagagct
     7321 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     7381 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     7441 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     7501 tgaatggcga atggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     7561 ttaaatcagc tcatttttta accaataggc cgtactgcga tgagtggcag ggcggggcgt
     7621 aattttttta aggcagttat tggtgccctt aaacgcctgg ttgctacgcc tgaataagtg
     7681 ataataagcg gatgaatggc agaaattcga aagcaaattc gacccggtcg tcggttcagg
     7741 gcagggtcgt taaatagccg cttatgtcta ttgctggttt accggtttat tgactaccgg
     7801 aagcagtgtg accgtgtgct tctcaaatgc ctgaggccag tttgctcagg ctctccccgt
     7861 ggaggtaata attgacgata tgatcattta ttctgcctcc cagagcctga taaaaacggt
     7921 gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg gctccatgca
     7981 ccgcgacgca acgcggggag gcagacaagg tatagggcgg cgaggcggct acagccgata
     8041 gtctggaaca gcgcacttac gggttgctgc gcaacccaag tgctaccggc gcggcagcgt
     8101 gacccgtgtc ggcggctcca acggctcgcc atcgtccaga aaacacggct catcgggcat
     8161 cggcaggcgc tgctgcccgc gccgttccca ttcctccgtt tcggtcaagg ctggcaggtc
     8221 tggttccatg cccggaatgc cgggctggct gggcggctcc tcgccggggc cggtcggtag
     8281 ttgctgctcg cccggataca gggtcgggat gcggcgcagg tcgccatgcc ccaacagcga
     8341 ttcgtcctgg tcgtcgtgat caaccaccac ggcggcactg aacaccgaca ggcgcaactg
     8401 gtcgcggggc tggccccacg ccacgcggtc attgaccacg taggccgaca cggtgccggg
     8461 gccgttgagc ttcacgacgg agatccagcg ctcggccacc aagtccttga ctgcgtattg
     8521 gaccgtccgc aaagaacgtc cgatgagctt ggaaagtgtc ttctggctga ccaccacggc
     8581 gttctggtgg cccatctgcg ccacgaggtg atgcagcagc attgccgccg tgggtttcct
     8641 cgcaataagc ccggcccacg cctcatgcgc tttgcgttcc gtttgcaccc agtgaccggg
     8701 cttgttcttg gcttgaatgc cgatttctct ggactgcgtg gccatgctta tctccatgcg
     8761 gtaggggtgc cgcacggttg cggcaccatg cgcaatcagc tgcaactttt cggcagcgcg
     8821 acaacaatta tgcgttgcgt aaaagtggca gtcaattaca gattttcttt aacctacgca
     8881 atgagctatt gcggggggtg ccgcaatgag ctgttgcgta cccccctttt ttaagttgtt
     8941 gatttttaag tctttcgcat ttcgccctat atctagttct ttggtgccca aagaagggca
     9001 cccctgcggg gttcccccac gccttcggcg cggctccccc tccggcaaaa agtggcccct
     9061 ccggggcttg ttgatcgact gcgcggcctt cggccttgcc caaggtggcg ctgccccctt
     9121 ggaacccccg cactcgccgc cgtgaggctc ggggggcagg cgggcgggct tcgcccttcg
     9181 ac
//
