LOCUS       sequence-214310-        6927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  (+)-Limonene Synthase from Citrus sinensis in pET-28a(+). N-terminal
            was truncated to remove amino acids 1-52 and then His-tagged for 
            purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6927)
  AUTHORS   Morehouse BR, Kumar RP, Matos JO, Olsen SN, Entova S, Oprian DD
  TITLE     Functional and Structural Characterization of a (+)-Limonene 
            Synthase from Citrus sinensis.
  JOURNAL   Biochemistry. 2017 Mar 28;56(12):1706-1715. doi: 
            10.1021/acs.biochem.7b00143. Epub 2017 Mar 15.
  PUBMED    28272875
REFERENCE   2  (bases 1 to 6927)
  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"; doi: 
            "10.1021/acs.biochem.7b00143"; journalName: "Biochemistry"; date: 
            "2017-03-28- 28"; volume: "56"; issue: "12"; pages: "1706-1715"
COMMENT     Sequence Label: (+)-Limonene Synthase
FEATURES             Location/Qualifiers
     source          1..6927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      45..633
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     534..553
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     787..804
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    819..958
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(884..903)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1044..1066
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1060..1251)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2060..3142)
                     /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     3104..3123
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3143..3220)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3426..3445
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3529..3548
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3529..3547
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3548..3572
                     /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             3587..3609
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3619..3636
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5320..5337
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5390..5408)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5404..5451
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5488..5943
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5575..5594)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5785..5806
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6035..6850)
                     /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(6035..6850)
                     /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     6758..6777
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6842..6861
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
       61 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
      121 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
      181 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
      241 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
      301 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
      361 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
      421 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
      481 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
      541 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
      601 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
      661 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
      721 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
      781 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
      841 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
      901 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
      961 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     1021 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     1081 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     1141 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     1201 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     1261 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     1321 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     1381 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     1441 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     1501 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     1561 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     1621 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     1681 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     1741 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     1801 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     1861 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     1921 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     1981 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     2041 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     2101 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     2161 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     2221 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     2281 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     2341 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     2401 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     2461 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     2521 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     2581 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     2641 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     2701 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     2761 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     2821 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     2881 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     2941 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     3001 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     3061 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     3121 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     3181 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     3241 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     3301 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     3361 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     3421 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     3481 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     3541 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
     3601 agaaggagat ataccatgca tcatcatcat catcatagga gatcagcaaa ctaccaacct
     3661 tcaatttggg accatgattt tttgcagtca ctgaatagca actatacgga tgaaacatac
     3721 aaaagacgag cagaagagct gaagggaaaa gtgaagacag cgattaagga tgtaaccgag
     3781 cctctggatc agttggagct gattgataat ttgcaaagac ttggattggc ttatcatttt
     3841 gagcctgaga ttcggaacat attgcgtaat atccacaacc ataataaaga ttataattgg
     3901 agaaaagaaa atctgtatgc aacctccctt gaattcagac tacttagaca acatggctat
     3961 cctgtttctc aagaggtttt cagtggtttt aaagacgaca aggtaggctt catttgtgat
     4021 gatttcaagg gaatactgag cttgcatgaa gcctcgtatt acagcttaga aggagaaagc
     4081 atcatggagg aggcctggca attcaccagt aagcatctta aagaaatgat gatcaccagc
     4141 aacagcaagg aagaggatgt atttgtagca gaacaagcga agcgggcgct ggagctccct
     4201 ctgcattgga aagcgcctat gttagaggca aggtggttca tacacgttta tgagaaaaga
     4261 gaggacaaga accacctttt acttgagctc gctaagttgg agtttaacac tttgcaggca
     4321 atttaccagg aagaacttaa agacatttca gggtggtgga aggatacagg tcttggagag
     4381 aaattgagct ttgcgaggaa caggttggta gcgtccttct tatggagcat ggggatcgcg
     4441 tttgagcctc aattcgccta ctgcaggaga gtgctcacaa tctcgatagc cctaattaca
     4501 gtgattgatg acatttatga tgtctatgga acattggatg aacttgagat attcactgat
     4561 gctgttgcga ggtgggacat caattatgcc ttgaagcacc ttccgggcta tatgaaaatg
     4621 tgttttcttg ccctttacaa ctttgttaat gaatttgctt attacgttct caaacaacag
     4681 gattttgata tgcttctgag cataaaacat gcatggcttg gcttaataca agcctacttg
     4741 gtggaggcga aatggtacca tagcaagtac acaccgaaac tggaagaata cttggaaaat
     4801 ggattggtat caataacggg ccctttaatt ataacgattt catatctttc tggtacaaat
     4861 ccaatcatta agaaggaact ggaatttcta gaaagtaatc cagatatagt tcactggtca
     4921 tccaagattt tccgtctgca agatgatttg ggaacttcat cggacgagat acagagaggg
     4981 gatgttccga aatcaatcca gtgttacatg catgaaactg gtgcctcgga ggaagttgct
     5041 cgtgaacaca tcaaggatat gatgagacag atgtggaaga aggtgaatgc atacacagcc
     5101 gataaagact ctcccttgac tcgaacaact gctgagttcc tcttgaatct tgtgcgaatg
     5161 tcccatttta tgtatctaca tggagatggg catggtgttc aaaaccaaga gactatcgat
     5221 gtcggcttta cattgctttt tcagcccatt cccttggagg acaaagacat ggctttcaca
     5281 gcatctcctg gcaccaaagg ctgagcggcc gcactcgagc accaccacca ccaccactga
     5341 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     5401 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     5461 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     5521 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     5581 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     5641 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     5701 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5761 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     5821 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     5881 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     5941 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     6001 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     6061 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     6121 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     6181 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     6241 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     6301 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     6361 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     6421 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     6481 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     6541 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     6601 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     6661 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     6721 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     6781 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     6841 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     6901 accaaaatcc cttaacgtga gttttcg
//
