LOCUS       62056_1930        8946 bp DNA     circular SYN 17-NOV-2021
DEFINITION  Expression vector p2BBAD-phaB2-phaC2, complete sequence.
ACCESSION   OL331254
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8946)
  AUTHORS   Tang R, Peng X, Weng C, Han Y.
  TITLE     The over-expression of phasin and regulator genes promoting the 
            synthesis of polyhydroxybutyrate in Cupriavidus necator H16 under 
            non-stress conditions
  JOURNAL   Appl Environ Microbiol, AEM0145821 (2021) In press
  PUBMED    34731058
REFERENCE   2  (bases 1 to 8946)
  AUTHORS   Tang R, Peng X, Weng C, Han Y.
  TITLE     Direct Submission
  JOURNAL   Submitted (31-OCT-2021) National Key Laboratory of Biochemical 
            Engineering, Institute of Process Engineering, Chinese Academy of 
            Sciences, 1 North 2nd Street, Zhongguancun PR, Beijing, Beijing 
            100190, China
REFERENCE   3  (bases 1 to 8946)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Appl 
            Environ Microbiol, AEM0145821 (2021) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (31-OCT-2021) National Key Laboratory of Biochemical Engineering, 
            Institute of Process Engineering, Chinese Academy of Sciences, 1 
            North 2nd Street, Zhongguancun PR, Beijing, Beijing 100190, China"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..8946
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1023..1792
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             1793..2452
                     /label=pBBR1 Rep
                     /note="replication protein for the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica"
     primer_bind     3162..3178
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3188..3206
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(3248..4123)
                     /label=araC
                     /note="L-arabinose regulatory protein"
     promoter        4150..4434
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     regulatory      4492..4507
                     /regulatory_class="ribosome_binding_site"
     gene            4514..5260
                     /gene="phaB2"
                     /label=phaB2
     CDS             4514..5260
                     /codon_start=1
                     /transl_table=11
                     /gene="phaB2"
                     /product="PhaB2"
                     /EC_number="1.1.1.36"
                     /label=phaB2
                     /note="acetoacetyl-CoA reductase; derived from Cupriavidus 
                     necator H16"
                     /protein_id="UEP67409.1"
                     /translation="MAGQRIALVTGGMGGLGEAIAVRLLADGARVVVTHSVHNDHVAQW
                     LGTQRSAGREFTAFPVDVTDFASCQRCVSQVRSELGDVDILINNAGVTRDRTLRKMDKA
                     DWDFVLRTDLDSLFHMTRPLVEPMLARGWGRIVNISSVNASRGAFGQTNYAAAKAGVHG
                     FTKALALELARKGITVNTVSPGYLDTHMVTDMPAEILERDVLPTIPVGRLGKPAEVAAL
                     ISYLCSDDGAFVTGANFAINGGQHLQ"
     gene            5261..7036
                     /gene="phaC2"
                     /label=phaC2
     CDS             5261..7036
                     /codon_start=1
                     /transl_table=11
                     /gene="phaC2"
                     /product="PhaC2"
                     /label=phaC2
                     /note="poly(3-hydroxybutyrate) polymerase; derived from 
                     Cupriavidus necator H16"
                     /protein_id="UEP67410.1"
                     /translation="MRIDDRTSPPSLSAMPECAPQAAIPAQVWMDKQIRAGLARATGGL
                     SVISASLAGLDWALQLGISPAKIVQAWQVWLQHSIEDWPRLLPTAAEPPSVDLSDQRFA
                     DAGWQDWPYRCWRDAFMRNEAFWESLTQDIDGLSRHHQHIVAFCARQWLDLLAPGNVWW
                     MNPTIARTASSTFGANFLQGAHHWLQDASELLSKVPGLPAGRPALEFLPGRDVARTPGK
                     VVFRNALIELIEYAPALGANTVWREPVLIVPSWIMRYYILDLKPEDSLVRYLVESGHTV
                     FMISWKNPDASARDFGLDTYLEAGLLTALNTVHARCDGAHVHAAGYCLGGTLLATGAAM
                     LARDAAGGPLASMTLFASETDFHDPGELGLFIDKSSLATLDALMWSQGYLDGPQMKSAF
                     QMLNAQDLIWSRVMSEYLLGQRLRANDLVSWNRDTTRLPYRLHSECLHKLFLGNELATG
                     KLCVGGQPVALSDLDLPLFVVGTEHDHVSPWRSVYKLHLLTKAELTFLLTSGGHNAGIV
                     SEPGHPGRRYHVHTRLPAAPYLSPEQFLNVAVYRDGSWWPAWHDWLVAHSTARCTRQAP
                     GQGLADAPGTYVLEP"
     terminator      7047..7093
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     promoter        complement(7137..7155)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7176..7192)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(7200..7216)
                     /label=lac operator
                     /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)."
     promoter        complement(7224..7254)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(7269..7290)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(7698..8489)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
ORIGIN
        1 ctcgggccgt ctcttgggct tgatcggcct tcttgcgcat ctcacgcgct cctgcggcgg
       61 cctgtagggc aggctcatac ccctgccgaa ccgcttttgt cagccggtcg gccacggctt
      121 ccggcgtctc aacgcgcttt gagattccca gcttttcggc caatccctgc ggtgcatagg
      181 cgcgtggctc gaccgcttgc gggctgatgg tgacgtggcc cactggtggc cgctccaggg
      241 cctcgtagaa cgcctgaatg cgcgtgtgac gtgccttgct gccctcgatg ccccgttgca
      301 gccctagatc ggccacagcg gccgcaaacg tggtctggtc gcgggtcatc tgcgctttgt
      361 tgccgatgaa ctccttggcc gacagcctgc cgtcctgcgt cagcggcacc acgaacgcgg
      421 tcatgtgcgg gctggtttcg tcacggtgga tgctggccgt cacgatgcga tccgccccgt
      481 acttgtccgc cagccacttg tgcgccttct cgaagaacgc cgcctgctgt tcttggctgg
      541 ccgacttcca ccattccggg ctggccgtca tgacgtactc gaccgccaac acagcgtcct
      601 tgcgccgctt ctctggcagc aactcgcgca gtcggcccat cgcttcatcg gtgctgctgg
      661 ccgcccagtg ctcgttctct ggcgtcctgc tggcgtcagc gttgggcgtc tcgcgctcgc
      721 ggtaggcgtg cttgagactg gccgccacgt tgcccatttt cgccagcttc ttgcatcgca
      781 tgatcgcgta tgccgccatg cctgcccctc ccttttggtg tccaaccggc tcgacggggg
      841 cagcgcaagg cggtgcctcc ggcgggccac tcaatgcttg agtatactca ctagactttg
      901 cttcgcaaag tcgtgaccgc ctacggcggc tgcggcgccc tacgggcttg ctctccgggc
      961 ttcgccctgc gcggtcgctg cgctcccttg ccagcccgtg gatatgtgga cgatggccgc
     1021 gagcggccac cggctggctc gcttcgctcg gcccgtggac aaccctgctg gacaagctga
     1081 tggacaggct gcgcctgccc acgagcttga ccacagggat tgcccaccgg ctacccagcc
     1141 ttcgaccaca tacccaccgg ctccaactgc gcggcctgcg gccttgcccc atcaattttt
     1201 ttaattttct ctggggaaaa gcctccggcc tgcggcctgc gcgcttcgct tgccggttgg
     1261 acaccaagtg gaaggcgggt caaggctcgc gcagcgaccg cgcagcggct tggccttgac
     1321 gcgcctggaa cgacccaagc ctatgcgagt gggggcagtc gaaggcgaag cccgcccgcc
     1381 tgccccccga gcctcacggc ggcgagtgcg ggggttccaa gggggcagcg ccaccttggg
     1441 caaggccgaa ggccgcgcag tcgatcaaca agccccggag gggccacttt ttgccggagg
     1501 gggagccgcg ccgaaggcgt gggggaaccc cgcaggggtg cccttctttg ggcaccaaag
     1561 aactagatat agggcgaaat gcgaaagact taaaaatcaa caacttaaaa aaggggggta
     1621 cgcaacagct cattgcggca ccccccgcaa tagctcattg cgtaggttaa agaaaatctg
     1681 taattgactg ccacttttac gcaacgcata attgttgtcg cgctgccgaa aagttgcagc
     1741 tgattgcgca tggtgccgca accgtgcggc accctaccgc atggagataa gcatggccac
     1801 gcagtccaga gaaatcggca ttcaagccaa gaacaagccc ggtcactggg tgcaaacgga
     1861 acgcaaagcg catgaggcgt gggccgggct tattgcgagg aaacccacgg cggcaatgct
     1921 gctgcatcac ctcgtggcgc agatgggcca ccagaacgcc gtggtggtca gccagaagac
     1981 actttccaag ctcatcggac gttctttgcg gacggtccaa tacgcagtca aggacttggt
     2041 ggccgagcgc tggatctccg tcgtgaagct caacggcccc ggcaccgtgt cggcctacgt
     2101 ggtcaatgac cgcgtggcgt ggggccagcc ccgcgaccag ttgcgcctgt cggtgttcag
     2161 tgccgccgtg gtggttgatc acgacgacca ggacgaatcg ctgttggggc atggcgacct
     2221 gcgccgcatc ccgaccctgt atccgggcga gcagcaacta ccgaccggcc ccggcgagga
     2281 gccgcccagc cagcccggca ttccgggcat ggaaccagac ctgccagcct tgaccgaaac
     2341 ggaggaatgg gaacggcgcg ggcagcagcg cctgccgatg cccgatgagc cgtgttttct
     2401 ggacgatggc gagccgttgg agccgccgac acgggtcacg ctgccgcgcc ggtagcactt
     2461 gggttgcgca gcaacccgta agtgcgctgt tccagactat cggctgtagc cgcctcgccg
     2521 ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga
     2581 cctgaatgga agccggcggc acctcgctaa cggattcacc gtttttatca ggctctggga
     2641 ggcagaataa atgatcatat cgtcaattat tacctccacg gggagagcct gagcaaactg
     2701 gcctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa
     2761 ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcgaa
     2821 tttgctttcg aatttctgcc attcatccgc ttattatcac ttattcaggc gtagcaccag
     2881 gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc cccgccctgc cactcatcgc
     2941 agtcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3001 aaatattaac gcttacaatt tccattcgcc attcaggctg cgcaactgtt gggaagggcg
     3061 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     3121 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
     3181 gcgcgcgtaa tacgactcac tatagggcga attggagctc caccgcggtg gcggccgctc
     3241 tagattatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact
     3301 cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa
     3361 aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg
     3421 cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga
     3481 aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa
     3541 aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca
     3601 tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca
     3661 gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc
     3721 caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg
     3781 caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc
     3841 actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg
     3901 ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc
     3961 cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa
     4021 aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa
     4081 acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccgc
     4141 cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt
     4201 actggctctt ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa
     4261 gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag
     4321 tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa
     4381 gattagcgga ttctacctga cgctttttat cgcaactctc tactgtttct ccatagggag
     4441 tccacaacgg tttccctcta gaaataattt tggaattcaa aagatctttt aagaaggaga
     4501 tatacataag cttatggccg gacaacgcat tgccctggta acgggaggca tgggcggcct
     4561 cggcgaagcg attgccgtgc ggctgctggc cgacggcgcg cgcgtcgtgg tgacgcactc
     4621 cgtacacaat gaccacgtgg cccagtggct cggcacgcaa cgctccgccg gccgcgaatt
     4681 cacggcgttc ccggtcgacg tgacggactt cgcgtcctgc cagcgctgcg tgtcgcaagt
     4741 gcgcagcgag ctcggcgacg tcgatattct gatcaacaat gccggggtca cgcgcgaccg
     4801 gaccctgcgc aagatggata aggctgactg ggacttcgtg ctgcgcacgg acctcgactc
     4861 gctctttcat atgacgcggc cgctggttga gccgatgttg gcgcgcggtt ggggccggat
     4921 cgtcaatatt tcttcggtca atgcctcgcg gggcgccttt ggccagacca actatgccgc
     4981 cgccaaggcg ggggtacatg gcttcaccaa ggcgctggcg ctggaactcg cacgcaaagg
     5041 catcaccgtc aacaccgttt cgccgggcta cctggatacg cacatggtga cggacatgcc
     5101 cgccgagatc ctcgagcggg acgtgttgcc gactatcccg gtcggccggc taggcaagcc
     5161 ggccgaggtc gcggcgctga tctcttacct gtgcagcgac gacggggcct tcgtcactgg
     5221 cgcgaatttt gccatcaacg gcgggcaaca cctgcaatga atgaggatcg acgacaggac
     5281 gtcaccgccc agcctgtcgg ccatgccgga atgcgcgccg caggccgcca taccggcgca
     5341 agtctggatg gacaagcaga tccgcgccgg cctggcccgt gccaccgggg gcctgtcggt
     5401 gatctcggca agtctggccg gactcgactg ggcgctgcag ctcggcatct ccccggcaaa
     5461 gattgtgcag gcatggcagg tgtggctgca gcacagcatc gaggactggc cacggctgtt
     5521 gccgaccgcg gccgagccgc cgtccgttga cctcagcgat cagcgctttg ccgatgccgg
     5581 ctggcaggac tggccttatc gctgctggcg cgatgcgttc atgcgcaacg aagccttctg
     5641 ggagtcgctg acgcaggata ttgacggcct gtcgcgtcat caccagcaca tcgtcgcctt
     5701 ctgcgcccgg caatggcttg acctgctcgc gcccggcaat gtttggtgga tgaatccgac
     5761 cattgccagg accgcctcga gcaccttcgg cgccaacttc ctgcaaggcg cccatcactg
     5821 gctgcaggac gccagtgagc tgttgtcaaa ggtgcctggg ctgcctgcgg ggcgccccgc
     5881 gctggaattt ctacccgggc gcgacgtggc gcgcacgcct ggcaaggtgg tgttccgcaa
     5941 cgcactaatt gaactgatcg agtatgcgcc cgcccttggc gccaatacag tctggcgcga
     6001 gccggtgctg atcgtgccgt cctggatcat gcggtactac atcctcgacc tgaagcccga
     6061 ggattcgctg gtgcgctacc tggtcgagag cggccatacc gtgttcatga tttcatggaa
     6121 gaacccggac gcctcggccc gcgacttcgg gcttgacacc tatctggagg ccgggcttct
     6181 gaccgcgctc aacaccgtgc acgcgcgctg cgacggcgcg cacgtccacg ccgccgggta
     6241 ttgcctgggc ggcaccctgc ttgccacggg tgccgccatg ctcgcccgcg acgccgccgg
     6301 cgggccactg gccagcatga cgctctttgc ctcagagacc gatttccatg atccgggcga
     6361 actgggcctg ttcatcgaca agagctcgct cgccacgctc gacgccctca tgtggagtca
     6421 gggatatctg gacgggcctc agatgaagtc tgcctttcag atgctgaacg cccaggacct
     6481 gatctggtcg cgcgtaatga gcgagtattt gctcggccaa cgtttgcgcg ccaacgatct
     6541 ggtctcctgg aaccgcgaca ccacccgctt gccttaccgc ctgcattcgg aatgcctgca
     6601 caagctgttc ctgggcaatg agctggccac cggcaagctg tgcgtgggcg gacagcccgt
     6661 cgccctgtcg gacctcgacc tcccgctttt cgtggttggc accgaacacg accacgtctc
     6721 gccctggcgc tccgtgtaca agctgcactt actgaccaag gctgaactga ccttcttgct
     6781 gacctcgggc ggccacaacg caggcatcgt gtccgagccg ggccacccgg gccggcgcta
     6841 ccatgtgcat acgcgattgc ccgccgcgcc ctacctgagc ccggagcaat tcctcaacgt
     6901 agctgtgtac cgcgacggtt cctggtggcc ggcgtggcat gactggctag tggcccactc
     6961 gacagcgcgc tgcacgcgac aggcgccggg ccaggggctc gcggacgctc ctggtaccta
     7021 cgtgctggaa ccctagaagc ttgcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     7081 cctttcgttt tatctgtgtc gacctcgagg gggggcccgg tacccagctt ttgttccctt
     7141 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     7201 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     7261 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     7321 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     7381 ttgcgtattg ggcgcatgca taaaaactgt tgtaattcat taagcattct gccgacatgg
     7441 aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct
     7501 tgcgtataat atttgcccat gggggtgggc gaagaactcc agcatgagat ccccgcgctg
     7561 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     7621 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa
     7681 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     7741 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     7801 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     7861 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     7921 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac
     7981 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     8041 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     8101 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     8161 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     8221 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     8281 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
     8341 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     8401 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     8461 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     8521 gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg
     8581 cagggcttcc caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc
     8641 cataaaaccg cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc
     8701 tttgcgcttg cgttttccct tgtccagata gcccagtagc tgacattcat cccaggtggc
     8761 acttttcggg gaaatgtgcg cgcccgcgtt cctgctggcg ctgggcctgt ttctggcgct
     8821 ggacttcccg ctgttccgtc agcagctttt cgcccacggc cttgatgatc gcggcggcct
     8881 tggcctgcat atcccgattc aacggcccca gggcgtccag aacgggcttc aggcgctccc
     8941 gaaggt
//
