LOCUS       sequence-81333-e        8707 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inserts the beta-phellandrene synthase gene and aadA resistance in 
            the rbcL-accD intergenic region in Nicociana tabacum .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8707)
  TITLE     Melis lab pNtCp-rbcL-accD
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8707)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pNtCp-rbcL-PHLS-aadA-accD
FEATURES             Location/Qualifiers
     source          1..8707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3874..4665
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     primer_bind     complement(6519..6539)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6519..6537)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6558..6574)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6558..6574)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6571..6593)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6582..6598
                     /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)."
     promoter        complement(join(6606..6612,6613..6630,6631..6636))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6651..6672
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6789..6806)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6960..7548)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7040..7059)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7719..8510,8511..8579))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8342..8361
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8580..8684)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcct
      661 tctactggta catggacaac tgtatggacc gatggactta ccagccttga tcgttacaaa
      721 gggcgatgct accgcatcga gcgtgttgtt ggagaaaaag atcaatatat tgcttatgta
      781 gcttaccctt tagacctttt tgaagaaggt tctgttacca acatgtttac ttccattgta
      841 ggtaacgtat ttgggttcaa agccctgcgc gctctacgtc tggaagatct gcgaatccct
      901 cctgcttatg ttaaaacttt ccaaggtccg cctcatggga tccaagttga aagagataaa
      961 ttgaacaagt atggtcgtcc cctgttggga tgtactatta aacctaaatt ggggttatct
     1021 gctaaaaact acggtagagc tgtttatgaa tgtcttcgcg gtggacttga ttttaccaaa
     1081 gatgatgaga acgtgaactc acaaccattt atgcgttgga gagatcgttt cttattttgt
     1141 gccgaagcac tttataaagc acaggctgaa acaggtgaaa tcaaagggca ttacttgaat
     1201 gctactgcag gtacatgcga agaaatgatc aaaagagctg tatttgctag agaattgggc
     1261 gttccgatcg taatgcatga ctacttaacg gggggattca ccgcaaatac tagcttggct
     1321 cattattgcc gagataatgg tctacttctt cacatccacc gtgcaatgca tgcggttatt
     1381 gatagacaga agaatcatgg tatccacttc cgggtattag caaaagcgtt acgtatgtct
     1441 ggtggagatc atattcactc tggtaccgta gtaggtaaac ttgaaggtga aagagacata
     1501 actttgggct ttgttgattt actgcgtgat gattttgttg aacaagatcg aagtcgcggt
     1561 atttatttca ctcaagattg ggtctcttta ccaggtgttc tacccgtggc ttcaggaggt
     1621 attcacgttt ggcatatgcc tgctctgacc gagatctttg gggatgattc cgtactacag
     1681 ttcggtggag gaactttagg acatccttgg ggtaatgcgc caggtgccgt agctaatcga
     1741 gtagctctag aagcatgtgt aaaagctcgt aatgaaggac gtgatcttgc tcaggaaggt
     1801 aatgaaatta ttcgcgaggc ttgcaaatgg agcccggaac tagctgctgc ttgtgaagta
     1861 tggaaagaga tcgtatttaa ttttgcagca gtggacgttt tggataagta aaaacagtag
     1921 acattagcag ataaattagc aggaaataaa gaaggataag gagaaagaac tcaagtaatt
     1981 atccttcgtt ctcttaattg aattgcaatt aaactcggcc caatctttta ctaaaaggat
     2041 tgagccgaat acaacaaacc cgggaagcta tcgctccccc gccgtcgttc aatgagaatg
     2101 gataagaggc tcgtgggatt gacgtgaggg ggcagggatg gctatatttc tgggagcgaa
     2161 ctccgggcga atacgaagcg cttggataca gttgtaggga gggatttatg tgtagtttgc
     2221 aagtttctga tcctattcct accggacgcc gttccggtgg ttatcccccg gccttatggg
     2281 atttcgatac tattcaatcc ctgaataccg aatataaggg cgaacgtcac atgcgtcggg
     2341 aagaagactt aattggtcaa gttcgggaaa tgttggtgca cgaagtagaa gatcccactc
     2401 cccagttgga attcattgac gatctgcata aattgggcat ttcctgccat tttgaaaacg
     2461 agattctgca aattctcaaa tccatttatc tcaaccaaaa ctataaacgg gacctctatt
     2521 ctaccagttt agccttccgt ctcttgcgtc aatacgggtt tatcttgccg caggaagttt
     2581 ttgactgctt taaaaacgaa gaaggtacgg attttaaacc cagcttcggc cgggatatta
     2641 agggtctgtt acagttgtac gaagcctcct ttttgtcccg gaagggggaa gaaactttac
     2701 aactcgcccg cgaatttgct accaaaatct tgcaaaagga agtcgatgaa cgggaatttg
     2761 ctactaaaat ggaatttccc agtcactgga ccgtacaaat gcctaacgct cggcctttta
     2821 tcgatgccta tcgtcggcgt cccgacatga accccgtggt tctggaactc gccattctcg
     2881 ataccaatat cgtgcaagct cagtttcaag aagaattgaa ggagacctcc cgttggtggg
     2941 aaagcacggg gattgttcaa gaactgccgt ttgttcggga ccggattgtg gaaggttatt
     3001 tttggaccat tggtgttact caacgccgtg aacacggtta cgaacgtatt atgacggcca
     3061 aagtcatcgc tttggtgacc tgtttggatg atatttatga cgtatatggc actattgaag
     3121 aattgcaact cttcacctct acgattcagc gttgggattt ggagtctatg aagcagttac
     3181 cgacttatat gcaggtaagc ttcctggcct tgcacaattt tgtaaccgaa gtggcctatg
     3241 atacgctgaa gaaaaagggc tacaactcta ccccctattt gcggaagact tgggtggatt
     3301 tggtcgaaag ttacattaag gaagccactt ggtactataa tgggtacaaa ccctctatgc
     3361 aggaatacct caacaacgcc tggatctctg tgggcagcat ggctattttg aatcatttgt
     3421 tttttcgctt tactaatgaa cgcatgcata agtaccggga catgaatcgt gtatcctcta
     3481 atattgtgcg gttagccgac gatatgggaa cctctttggc cgaagttgaa cgcggtgacg
     3541 tgcccaaagc tatccaatgt tacatgaatg aaacgaacgc ctctgaggag gaggcccgcg
     3601 aatatgtgcg gcgcgttatc caggaagaat gggaaaaact gaacactgaa ctgatgcgcg
     3661 acgacgacga tgacgatgat ttcaccttaa gtaaatacta ctgcgaagtc gttgctaacc
     3721 tgacccggat ggctcagttc atttaccaag atggttccga tgggtttggg atgaaagatt
     3781 ccaaagtaaa tcgtttactg aaagaaacgc tgattgagcg ctatgagtga gcggccgcac
     3841 gaagcgcttg gatacagttg tagggaggga tttatgaggg aagcggtgat cgccgaagta
     3901 tcgactcaac tatcagaggt agttggcgtc atcgagcgcc atctcgaacc gacgttgctg
     3961 gccgtacatt tgtacggctc cgcagtggat ggcggcctga agccacacag tgatattgat
     4021 ttgctggtta cggtgaccgt aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac
     4081 cttttggaaa cttcggcttc ccctggagag agcgagattc tccgcgctgt agaagtcacc
     4141 attgttgtgc acgacgacat cattccgtgg cgttatccag ctaagcgcga actgcaattt
     4201 ggagaatggc agcgcaatga cattcttgca ggtatcttcg agccagccac gatcgacatt
     4261 gatctggcta tcttgctgac aaaagcaaga gaacatagcg ttgccttggt aggtccagcg
     4321 gcggaggaac tctttgatcc ggttcctgaa caggatctat ttgaggcgct aaatgaaacc
     4381 ttaacgctat ggaactcgcc gcccgactgg gctggcgatg agcgaaatgt agtgcttacg
     4441 ttgtcccgca tttggtacag cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc
     4501 gactgggcaa tggagcgcct gccggcccag tatcagcccg tcatacttga agctagacag
     4561 gcttatcttg gacaagaaga agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt
     4621 gtccactacg tgaaaggcga gatcaccaag gtagtcggca aataataaga tcttggccta
     4681 gtctatagga ggttttgaaa agaaaggagc aataatcatt ttcttgttct atcaagaggg
     4741 tgctattgct cctttctttt tttcttttta tttatttact agtattttac ttacatagac
     4801 ttttttgttt acattataga aaaagaagga gaggttattt tcttgcattt attcatgatt
     4861 gagtattcta ttttgatttt gtatttgttt aaattgtgaa atagaacttg tttctcttct
     4921 tgctaatgtt actatatctt tttgattttt tttttccaaa aaaaaaatca aattttgact
     4981 tcttcttatc tcttatcttt gaatatctct tatctttgaa ataataatat cattgaaata
     5041 agaaagaaga gctatattcg aacttgaatc ttttgttttc taatttaaat aatgtaaaaa
     5101 cggaatgtaa gtacccgggg attctattgc atatattttg actaagtata tacttaccta
     5161 gatatacaag atttgaaata caaaatctag aaaactaaat caaaatctaa gactcaaatc
     5221 tttctattgt tgtcttggat ccacaattaa tcctacggat ccttaggatt ggtatattct
     5281 tttctatcct gtagtttgta gtttccctga atcaagccaa gtatcacacc tctttctacc
     5341 catcctgtac attgtcccct ttgttccgtg ttgaaataga accttaattt attacttatt
     5401 tttttattaa attttagatt tgttagtgat tagatattag tattagacga gattttacga
     5461 aacaattatt tttttatttc tttataggag aggacaaatc tcttttttcg atgcgaattt
     5521 gacacgacat aggagaagcc gccctttatt aaaaattata ttattttaaa taatataaag
     5581 ggggttccaa catattaata tatagtgaag tgttccccca gattcagaac tttttttcaa
     5641 tactcacaat ccttattagt taataatcct agtgattgga tttctatgct tagtctgata
     5701 ggaaataaga tattcaaata aataatttta tagcgaatga ctattcatct attgtatttt
     5761 catgcaaata gggggcaaga aaactctatg gaaagatggt ggtttaattc gatgttgttt
     5821 aagaaggagt tcgaacgcag gtgtgggcta aataaatcaa tgggcagtct tggtcctatt
     5881 gaaaatacca atgaagatcc aaatcgaaaa gtgaaaaaca ttcatagttg gaggaatcgt
     5941 gacaattcta gttgcagtaa tgttgattat ttattcggcg ttaaagacat tcggaatttc
     6001 atctctgatg acactttttt agttagtgat aggaatggag acagttattc catctatttt
     6061 gatattgaaa atcatatttt tgagattgac aacgatcatt cttttctgag tgaactagaa
     6121 agttcttttt atagttatcg aaactcgaat tatcggaata atggatttag gggcgaagat
     6181 ccctactata attcttacat gtatgatact caatatagtt ggaataatca cattaatagt
     6241 tgcattgata gttaccttca gtctcaaatc tgtatagata cttccattat aagtggtagt
     6301 gagaattacg gtgacagtta catttatagg gccgtttgtg gtggtgaaag tcgaaatagt
     6361 agtgaaaacg agggttccag tagacgaact cgcacgaagg gcagtgattt aactataaga
     6421 gaaagttcta atgatctcga ggtaactcaa aaatacaggc atttgtgggt tcaatgcgag
     6481 tcgacctcga gggggggccc ggtacccagc ttttgttccc tttagtgagg gttaattgcg
     6541 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     6601 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     6661 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6721 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     6781 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     6841 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     6901 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6961 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     7021 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     7081 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     7141 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     7201 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     7261 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     7321 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     7381 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     7441 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     7501 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     7561 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     7621 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     7681 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     7741 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     7801 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     7861 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     7921 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     7981 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     8041 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     8101 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     8161 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     8221 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     8281 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     8341 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     8401 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     8461 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     8521 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     8581 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     8641 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     8701 gtgccac
//
