LOCUS       sequence-81334-e        9715 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inserts the beta-phellandrene synthase gene, the geranyl diphosphate
            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 9715)
  TITLE     Melis lab pNtCp-rbcL-accD
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9715)
  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            "Unpublished"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-GPPS-aadA-accD
FEATURES             Location/Qualifiers
     source          1..9715
                     /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             4882..5673
                     /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(7527..7547)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(7527..7545)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7566..7582)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7566..7582)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7579..7601)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7590..7606
                     /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(7614..7620,7621..7638,7639..7644))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7659..7680
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7797..7814)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7968..8556)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8048..8067)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8727..9518,9519..9587))
                     /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     9350..9369
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9588..9692)
                     /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 gcggcctcga
     3841 ctaacactgt ataacattaa gaaggaggta aaatgagctc cacccggtct agtaaagcgt
     3901 tagttcaact ggcggatttg tccgaacagg tgaaaaacgt ggtggagttt gattttgaca
     3961 aatacatgca tagtaaggcg attgcggtta atgaagcctt agataaggtt atcccccccc
     4021 gttacccgca gaaaatttat gagagcatgc ggtacagttt gctagccggc gggaaacgag
     4081 tgcgtccaat tttatgtatt gcggcctgcg aattgatggg cggtaccgaa gagttggcca
     4141 tgcccactgc ttgtgccatt gaaatgattc ataccatgtc cctcattcat gatgacttac
     4201 cctacattga taatgatgat ctccgccgag gtaaacccac caatcacaaa gttttcgggg
     4261 aggataccgc tattattgcc ggagatgctt tattgagctt agcatttgaa catgtggcgg
     4321 tctccaccag ccggactctg gggacggata ttattttgcg cctattaagc gaaatcggac
     4381 gggcaactgg gtccgaaggt gtcatgggcg ggcaagtggt tgatattgag agtgaaggcg
     4441 acccttctat tgatttagag accttagaat gggtgcacat tcataaaact gcagtattgt
     4501 tagaatgttc tgtcgtgtgt ggcgctatca tgggaggagc ctcagaagat gacattgaac
     4561 gcgcccggcg ctatgcgcga tgcgtaggcc tactctttca agtagtggat gatattcttg
     4621 atgtgagcca gtccagtgaa gagttgggga aaaccgccgg taaagactta atttccgata
     4681 aagctaccta ccccaaatta atgggtctgg aaaaagcgaa agagttcgca gatgaattgc
     4741 tcaacagggg taagcaagaa ttatcatgct ttgatcctac taaagctgct cccctttttg
     4801 ctttggccga ctatattgct agtcgccaga attaactcga ggccgcacga agcgcttgga
     4861 tacagttgta gggagggatt tatgagggaa gcggtgatcg ccgaagtatc gactcaacta
     4921 tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc cgtacatttg
     4981 tacggctccg cagtggatgg cggcctgaag ccacacagtg atattgattt gctggttacg
     5041 gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga tcaacgacct tttggaaact
     5101 tcggcttccc ctggagagag cgagattctc cgcgctgtag aagtcaccat tgttgtgcac
     5161 gacgacatca ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg agaatggcag
     5221 cgcaatgaca ttcttgcagg tatcttcgag ccagccacga tcgacattga tctggctatc
     5281 ttgctgacaa aagcaagaga acatagcgtt gccttggtag gtccagcggc ggaggaactc
     5341 tttgatccgg ttcctgaaca ggatctattt gaggcgctaa atgaaacctt aacgctatgg
     5401 aactcgccgc ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt
     5461 tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg
     5521 gagcgcctgc cggcccagta tcagcccgtc atacttgaag ctagacaggc ttatcttgga
     5581 caagaagaag atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg
     5641 aaaggcgaga tcaccaaggt agtcggcaaa taataagatc ttggcctagt ctataggagg
     5701 ttttgaaaag aaaggagcaa taatcatttt cttgttctat caagagggtg ctattgctcc
     5761 tttctttttt tctttttatt tatttactag tattttactt acatagactt ttttgtttac
     5821 attatagaaa aagaaggaga ggttattttc ttgcatttat tcatgattga gtattctatt
     5881 ttgattttgt atttgtttaa attgtgaaat agaacttgtt tctcttcttg ctaatgttac
     5941 tatatctttt tgattttttt tttccaaaaa aaaaatcaaa ttttgacttc ttcttatctc
     6001 ttatctttga atatctctta tctttgaaat aataatatca ttgaaataag aaagaagagc
     6061 tatattcgaa cttgaatctt ttgttttcta atttaaataa tgtaaaaacg gaatgtaagt
     6121 acccggggat tctattgcat atattttgac taagtatata cttacctaga tatacaagat
     6181 ttgaaataca aaatctagaa aactaaatca aaatctaaga ctcaaatctt tctattgttg
     6241 tcttggatcc acaattaatc ctacggatcc ttaggattgg tatattcttt tctatcctgt
     6301 agtttgtagt ttccctgaat caagccaagt atcacacctc tttctaccca tcctgtacat
     6361 tgtccccttt gttccgtgtt gaaatagaac cttaatttat tacttatttt tttattaaat
     6421 tttagatttg ttagtgatta gatattagta ttagacgaga ttttacgaaa caattatttt
     6481 tttatttctt tataggagag gacaaatctc ttttttcgat gcgaatttga cacgacatag
     6541 gagaagccgc cctttattaa aaattatatt attttaaata atataaaggg ggttccaaca
     6601 tattaatata tagtgaagtg ttcccccaga ttcagaactt tttttcaata ctcacaatcc
     6661 ttattagtta ataatcctag tgattggatt tctatgctta gtctgatagg aaataagata
     6721 ttcaaataaa taattttata gcgaatgact attcatctat tgtattttca tgcaaatagg
     6781 gggcaagaaa actctatgga aagatggtgg tttaattcga tgttgtttaa gaaggagttc
     6841 gaacgcaggt gtgggctaaa taaatcaatg ggcagtcttg gtcctattga aaataccaat
     6901 gaagatccaa atcgaaaagt gaaaaacatt catagttgga ggaatcgtga caattctagt
     6961 tgcagtaatg ttgattattt attcggcgtt aaagacattc ggaatttcat ctctgatgac
     7021 acttttttag ttagtgatag gaatggagac agttattcca tctattttga tattgaaaat
     7081 catatttttg agattgacaa cgatcattct tttctgagtg aactagaaag ttctttttat
     7141 agttatcgaa actcgaatta tcggaataat ggatttaggg gcgaagatcc ctactataat
     7201 tcttacatgt atgatactca atatagttgg aataatcaca ttaatagttg cattgatagt
     7261 taccttcagt ctcaaatctg tatagatact tccattataa gtggtagtga gaattacggt
     7321 gacagttaca tttatagggc cgtttgtggt ggtgaaagtc gaaatagtag tgaaaacgag
     7381 ggttccagta gacgaactcg cacgaagggc agtgatttaa ctataagaga aagttctaat
     7441 gatctcgagg taactcaaaa atacaggcat ttgtgggttc aatgcgagtc gacctcgagg
     7501 gggggcccgg tacccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     7561 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     7621 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     7681 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     7741 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     7801 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     7861 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     7921 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     7981 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     8041 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     8101 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     8161 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     8221 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     8281 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     8341 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     8401 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     8461 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     8521 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     8581 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     8641 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     8701 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     8761 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     8821 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     8881 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     8941 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     9001 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     9061 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     9121 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     9181 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     9241 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     9301 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     9361 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     9421 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     9481 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     9541 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     9601 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     9661 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccac
//
