LOCUS       sequence-361465-        8415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP dropout for assembling a double-stranded RNA expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8415)
  AUTHORS   Lariviere PJ, Leonard SP, Horak RD, Powell JE, Barrick JE
  TITLE     Honey bee functional genomics using symbiont-mediated RNAi.
  JOURNAL   Nat Protoc. 2023 Mar;18(3):902-928. doi: 10.1038/s41596-022-00778-4.
            Epub 2022 Dec 2.
  PUBMED    36460809
REFERENCE   2  (bases 1 to 8415)
  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: 
            "10.1038/s41596-022-00778-4"; journalName: "Nat Protoc"; date: 
            "2023-03"; volume: "18"; issue: "3"; pages: "902-928"
COMMENT     Sequence Label: pBTK800
FEATURES             Location/Qualifiers
     source          1..8415
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            2091..2178
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     rep_origin      2520..2914
                     /label=RSF1010 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     RSF1010; requires the RSF1010 RepA/B/C proteins for 
                     replication (Scholz et al., 1989)"
     RBS             3831..3842
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3849..4562
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      4574..4645
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4661..4688
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(4937..5728)
                     /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"
     CDS             complement(6108..6959)
                     /codon_start=1
                     /gene="repC"
                     /product="replication protein C of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepC
                     /translation="MVKPKNKHSLSHVRHDPAHCLAPGLFRALKRGERKRSKLDVTYDY
                     GDGKRIEFSGPEPLGADDLRILQGLVAMAGPNGLVLGPEPKTEGGRQLRLFLEPKWEAV
                     TADAMVVKGSYRALAKEIGAEVDSGGALKHIQDCIERLWKVSIIAQNGRKRQGFRLLSE
                     YASDEADGRLYVALNPLIAQAVMGGGQHVRISMDEVRALDSETARLLHQRLCGWIDPGK
                     TGKASIDTLCGYVWPSEASGSTMRKRRQRVREALPELVALGWTVTEFAAGKYDITRPKA
                     AG"
     CDS             complement(6946..7785)
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein A of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepA
                     /translation="MATHKPINILEAFAAAPPPLDYVLPNMVAGTVGALVSPGGAGKSM
                     LALQLAAQIAGGPDLLEVGELPTGPVIYLPAEDPPTAIHHRLHALGAHLSAEERQAVAD
                     GLLIQPLIGSLPNIMAPEWFDGLKRAAEGRRLMVLDTLRRFHIEEENASGPMAQVIGRM
                     EAIAADTGCSIVFLHHASKGAAMMGAGDQQQASRGSSVLVDNIRWQSYLSSMTSAEAEE
                     WGVDDDQRRFFVRFGVSKANYGAPFADRWFRRHDGGVLKPAVLERQRKSKGVPRGEA"
     CDS             complement(join(8296..8415,1..852))
                     /codon_start=1
                     /gene="repB"
                     /product="replication protein B of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepB
                     /translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDATTGQMMNREWSAAEVL
                     QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
                     YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
                     EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
ORIGIN
        1 ctcgatgtaa tccgcttcgt ggccgggctt gcgctctgcc agcgctgggc tggcctcggc
       61 catggccttg ccgatttcct cggcactgcg gccccggctg gccagcttct gcgcggcgat
      121 aaagtcgcac ttgctgaggt catcaccgaa gcgcttgacc agcccggcca tctcgctgcg
      181 gtactcgtcc agcgccgtgc gccggtggcg gctaagctgc cgctcgggca gttcgaggct
      241 ggccagcctg cgggccttct cctgctgccg ctgggcctgc tcgatctgct ggccagcctg
      301 ctgcaccagc gccgggccag cggtggcggt cttgcccttg gattcacgca gcagcaccca
      361 cggctgataa ccggcgcggg tggtgtgctt gtccttgcgg ttggtgaagc ccgccaagcg
      421 gccatagtgg cggctgtcgg cgctggccgg gtcggcgtcg tactcgctgg ccagcgtccg
      481 ggcaatctgc ccccgaagtt caccgcctgc ggcgtcggcc accttgaccc atgcctgata
      541 gttcttcggg ctggtttcca ctaccagggc aggctcccgg ccctcggctt tcatgtcatc
      601 caggtcaaac tcgctgaggt cgtccaccag caccagacca tgccgctcct gctcggcggg
      661 cctgatatac acgtcattgc cctgggcatt catccgcttg agccatggcg tgttctggag
      721 cacttcggcg gctgaccatt cccggttcat catctggccg gtggtggcgt ccctgacgcc
      781 gatatcgaag cgctcacagc ccatggcctt gagctgtcgg cctatggcct gcaaagtcct
      841 gtcgttcttc atcgggccac caagcgcagc cagatcgagc cgtcctcggt tgtcagtggc
      901 gtcaggtcga gcaagagcaa cgatgcgatc agcagcacca ccgtaggcat catggaagcc
      961 agcatcacgg ttagccatag cttccagtgc cacccccgcg acgcgctccg ggcgctctgc
     1021 gcggcgctgc tcacctcggc ggctacctcc cgcaactctt tggccagctc cacccatgcc
     1081 gcccctgtct ggcgctgggc tttcagccac tccgccgcct gcgcctcgct ggcctgctgg
     1141 gtctggctca tgacctgccg ggcttcgtcg gccagtgtcg ccatgctctg ggccagcggt
     1201 tcgatctgct ccgctaactc gttgatgcct ctggatttct tcactctgtc gattgcgttc
     1261 atggtctatt gcctcccggt attcctgtaa gtcgatgatc tgggcgttgg cggtgtcgat
     1321 gttcagggcc acgtctgccc ggtcggtgcg gatgccccgg ccttccatct ccaccacgtt
     1381 cggccccagg tgaacaccgg gcaggcgctc gatgccctgc gcctcaagtg ttctgtggtc
     1441 aatgcgggcg tcgtggccag cccgctctaa tgcccggttg gcatggtcgg cccatgcctc
     1501 gcgggtctgc tcaagccatg ccttgggctt gagcgcttcg gtcttctgtg ccccgccctt
     1561 ctccggggtc ttgccgttgt accgcttgaa ccactgagcg gcgggccgct cgatgccgtc
     1621 attgatccgc tcggagatca tcaggtggca gtgcgggttc tcgccgccac cggcatggat
     1681 ggccagcgta tacggcaggc gctcggcacc ggtcaggtgc tgggcgaact cggacgccag
     1741 cgccttctgc tggtcgaggg tcagctcgac cggcagggca aattcgacct ccttgaacag
     1801 ccgcccattg gcgcgttcat acaggtcggc agcatcccag tagtcggcgg gccgctcgac
     1861 gaactccggc atgtgcccgg attcggcgtg caagacttca tccatgtcgc gggcatactt
     1921 gccttcgcgc tggatgtagt cggccttggc cctggccgat tggccgcccg acctgctgcc
     1981 ggttttcgcc gtaaggtgat aaatcgccat gctgcctcgc tgttgctttt gcttttcggc
     2041 tccatgcaat ggccctcgga gagcgcaccg cccgaagggt ggccgttagg ccagtttctc
     2101 gaagagaaac cggtaagtgc gccctcccct acaaagtagg gtcgggattg ccgccgctgt
     2161 gcctccatga tagcctacga gacagcacat taacaatggg gtgtcaagat ggttaagggg
     2221 agcaacaagg cggcggatcg gctggccaag ctcgaagaac aacgagcgcg aatcaatgcc
     2281 gaaattcagc gggtgcgggc aagggaacag cagcaagagc gcaagaacga aacaaggcgc
     2341 aaggtgctgg tgggggccat gattttggcc aaggtgaaca gcagcgagtg gccggaggat
     2401 cggctcatgg cggcaatgga tgcgtacctt gaacgcgacc acgaccgcgc cttgttcggt
     2461 ctgccgccac gccagaagga tgagccgggc tgaatgatcg accgagacag gccctgcggg
     2521 gctgcacacg cgcccccacc cttcgggtag ggggaaaggc cgctaaagcg gctaaaagcg
     2581 ctccagcgta tttctgcggg gtttggtgtg gggtttagcg ggctttgccc gcctttcccc
     2641 ctgccgcgca gcggtggggc ggtgtgtagc ctagcgcagc gaatagacca gctatccggc
     2701 ctctggccgg gcatattggg caagggcagc agcgccccac aagggcgctg ataaccgcgc
     2761 ctagtggatt attcttagat aatcatggat ggatttttcc aacaccccgc cagcccccgc
     2821 ccctgctggg tttgcaggtt tgggggcgtg acagttattg caggggttcg tgacagttat
     2881 tgcagggggg cgtgacagtt attgcagggg ttcgtgacag ttagtacggg agtgacgggc
     2941 actggctggc aatgtctagc aacggcaggc atttcggctg agggtaaaag aactttccgc
     3001 taagcgatag actgtatgta aacacagtat tgcaaggacg cggaacatgc ctcatgtggc
     3061 ggccaggacg gccagccggg atcgggatac tggtcgttac cagagccacc gacccgagca
     3121 aacccttctc tatcagatcg ttgacgagta ttacccggca ttcgctgcgc ttatggcaga
     3181 gcagggaaag gaattgccgg gctatgtgca acgggaattt gaagaatttc tccaatgcgg
     3241 gcggctggag catggctttc tacgggttcg ctgcgagtct tgccacgccg agcacctggt
     3301 cgctttcaga aatcaatcta aagtatatat gagtaaactt ggtctgacag gcccctgaat
     3361 tcgcatctag actgatgaga cgtggtagag ccacaaacag ccggtacaag caacgatctc
     3421 caggaccatc tgaatcatgc gcggatgaca cgaactcacg acggcgatca cagacattaa
     3481 cccacagtac agacactgcg acaacgtggc aattcgtcgc aatacaacgg acagtcattc
     3541 atctttctgc ccctccaaaa gcaaaaaccc gccgaagcgg gtttttacgt aaatcaggtg
     3601 aaactgaccg actttggcag tttattcttg acatgtagtg agggggctgg tataatcaca
     3661 tagtactgtt ttatgtgaga ccgaaagtga aacgtgattt catgcgtcat tttgaacatt
     3721 ttgtaaatct tatttaataa tgtgtgcggc aattcacatt taatttatga atgttttctt
     3781 aacatcgcgg caactcaaga aacggcaggt tcggatctta gctactagag aaagaggaga
     3841 aatactagat gcgtaaaggc gaagagctgt tcactggtgt cgtccctatt ctggtggaac
     3901 tggatggtga tgtcaacggt cataagtttt ccgtgcgtgg cgagggtgaa ggtgacgcaa
     3961 ctaatggtaa actgacgctg aagttcatct gtactactgg taaactgccg gttccttggc
     4021 cgactctggt aacgacgctg acttatggtg ttcagtgctt tgctcgttat ccggaccata
     4081 tgaagcagca tgacttcttc aagtccgcca tgccggaagg ctatgtgcag gaacgcacga
     4141 tttcctttaa ggatgacggc acgtacaaaa cgcgtgcgga agtgaaattt gaaggcgata
     4201 ccctggtaaa ccgcattgag ctgaaaggca ttgactttaa agaggacggc aatatcctgg
     4261 gccataagct ggaatacaat tttaacagcc acaatgttta catcaccgcc gataaacaaa
     4321 aaaatggcat taaagcgaat tttaaaattc gccacaacgt ggaggatggc agcgtgcagc
     4381 tggctgatca ctaccagcaa aacactccaa tcggtgatgg tcctgttctg ctgccagaca
     4441 atcactatct gagcacgcaa agcgttctgt ctaaagatcc gaacgagaaa cgcgatcata
     4501 tggttctgct ggagttcgta accgcagcgg gcatcacgca tggtatggat gaactgtaca
     4561 aatgaccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
     4621 ctgttgtttg tcggtgaacg ctctctacta gagtcacact ggctcacctt cgggtgggcc
     4681 tttctgcgtt tataggtckc anatmmaaca gtactatgtg attataccag ccccctcact
     4741 acatgtcaag aataaactgc caaaggtaat cgttaatccg caaataacgt aaaaacccgc
     4801 ttcggcgggt ttttttatgg ggggagttta gggaaagagc atttgtcagc tgcgtctcaa
     4861 gcagttacag agatgttacg aacccccagg acatccgaga atgcgaggcg atggagggta
     4921 caacccgagc ggccgcttat ttgccgacta ccttggtgat ctcgcctttc acgtagtgga
     4981 caaattcttc caactgatct gcgcgcgagg ccaagcgatc ttcttcttgt ccaagataag
     5041 cctgtctagc ttcaagtatg acgggctgat actgggccgg caggcgctcc attgcccagt
     5101 cggcagcgac atccttcggc gcgattttgc cggttactgc gctgtaccaa atgcgggaca
     5161 acgtaagcac tacatttcgc tcatcgccag cccagtcggg cggcgagttc catagcgtta
     5221 aggtttcatt tagcgcctca aatagatcct gttcaggaac cggatcaaag agttcctccg
     5281 ccgctggacc taccaaggca acgctatgtt ctcttgcttt tgtcagcaag atagccagat
     5341 caatgtcgat cgtggctggc tcgaagatac ccgcaagaat gtcattgcgc tgccattctc
     5401 caaattgcag ttcgcgctta gctggataac gccacggaat gatgtcgtcg tgcacaacaa
     5461 tggtgacttc tacagcgcgg agaatctcgc tctctccagg ggaagccgaa gtttccaaaa
     5521 ggtcgttgat caaagctcgc cgcgttgttt catcaagcct tacggtcacc gtaaccagca
     5581 aatcaatatc actgtgtggc ttcaggccgc catccactgc ggagccgtac aaatgtacgg
     5641 ccagcaacgt cggttcgaga tggcgctcga tgacgccaac tacctctgat agttgagtcg
     5701 atacttcggc gatcaccgct tccctcataa tgtttaactt tgttttaggg cgactgccct
     5761 gctgcgtaac atcgttgctg ctccataaca tcaaacatcg acccacggcg taacgcgctt
     5821 gctgcttgga tgcccgaggc atagactgta ccccaaaaaa acagtcataa caagccatga
     5881 aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg
     5941 agcgcatacg ctacttgcat tacagcttac gaaccgaaca ggcttatgtc cactgggttc
     6001 gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggtagc agcgaagtcg
     6061 aggcatttct gtcctggctg gtcatgacca aaatccctta acgtgagtca gcctgccgcc
     6121 ttgggccggg tgatgtcgta cttgcccgcc gcgaactcgg ttaccgtcca gcccagcgcg
     6181 accagctccg gcaacgcctc gcgcacccgc tggcggcgct tgcgcatggt cgaaccactg
     6241 gcctctgacg gccagacata gccgcacaag gtatctatgg aagccttgcc ggttttgccg
     6301 gggtcgatcc agccacacag ccgctggtgc agcaggcggg cggtttcgct gtccagcgcc
     6361 cgcacctcgt ccatgctgat gcgcacatgc tggccgccac ccatgacggc ctgcgcgatc
     6421 aaggggttca gggccacgta caggcgcccg tccgcctcgt cgctggcgta ctccgacagc
     6481 agccgaaacc cctgccgctt gcggccattc tgggcgatga tggatacctt ccaaaggcgc
     6541 tcgatgcagt cctgtatgtg cttgagcgcc ccaccactat cgacctctgc cccgatttcc
     6601 tttgccagcg cccgatagct acctttgacc accatggcat cagcggtgac ggcctcccac
     6661 ttgggttcca ggaacagccg gagctgccgt ccgccttcgg tcttgggttc cgggccaagc
     6721 actaggccat taggcccagc catggccacc agcccttgca ggatgcgcag atcatcagcg
     6781 cccagcggct ccgggccgct gaactcgatc cgcttgccgt cgccgtagtc atacgtcacg
     6841 tccagcttgc tgcgcttgcg ctcgccccgc ttgagggcac ggaacaggcc gggggccaga
     6901 cagtgcgccg ggtcgtgccg gacgtggctg aggctgtgct tgttcttagg cttcaccacg
     6961 gggcaccccc ttgctcttgc gctgcctctc cagcacggcg ggcttgagca ccccgccgtc
     7021 atgccgcctg aaccaccgat cagcgaacgg tgcgccatag ttggccttgc tcacaccgaa
     7081 gcggacgaag aaccggcgct ggtcgtcgtc cacaccccat tcctcggcct cggcgctggt
     7141 catgctcgac aggtaggact gccagcggat gttatcgacc agtaccgagc tgccccggct
     7201 ggcctgctgc tggtcgcctg cgcccatcat ggccgcgccc ttgctggcat ggtgcaggaa
     7261 cacgatagag cacccggtat cggcggcgat ggcctccatg cgaccgatga cctgggccat
     7321 ggggccgctg gcgttttctt cctcgatgtg gaaccggcgc agcgtgtcca gcaccatcag
     7381 gcggcggccc tcggcggcgc gcttgaggcc gtcgaaccac tccggggcca tgatgttggg
     7441 caggctgccg atcagcggct ggatcagcag gccgtcagcc acggcttgcc gttcctcggc
     7501 gctgaggtgc gccccaaggg cgtgcaggcg gtgatgaatg gcggtgggcg ggtcttcggc
     7561 gggcaggtag atcaccgggc cggtgggcag ttcgcccacc tccagcagat ccggcccgcc
     7621 tgcaatctgt gcggccagtt gcagggccag catggattta ccggcaccac cgggcgacac
     7681 cagcgccccg accgtaccgg ccaccatgtt gggcaaaacg tagtccagcg gtggcggcgc
     7741 tgctgcgaac gcctccagaa tattgatagg cttatgggta gccattgatt gcctcctttg
     7801 caggcagttg gtggttaggc gctggcgggg tcactacccc cgccctgcgc cgctctgagt
     7861 tcttccaggc actcgcgcag cgcctcgtat tcgtcgtcgg tcagccagaa cttgcgctga
     7921 cgcatccctt tggccttcat gcgctcggca tatcgcgctt ggcgtacagc gtcagggctg
     7981 gccagcaggt cgccggtctg cttgtccttt tggtctttca tatcagtcac cgagaaactt
     8041 gccggggccg aaaggcttgt cttcgcggaa caaggacaag gtgcagccgt caaggttaag
     8101 gctggccata tcagcgactg aaaagcggcc agcctcggcc ttgtttgacg tataaccaaa
     8161 gccaccgggc aaccaatagc ccttgtcact tttgatcagg tagaccgacc ctgaagcgct
     8221 tttttcgtat tccataaaac ccccttctgt gcgtgagtac tcatagtata acaggcgtga
     8281 gtaccaacgc aagcactaca tgctgaaatc tggcccgccc ctgtccatgc ctcgctggcg
     8341 gggtgccggt gcccgtgcca gctcggcccg cgcaagctgg acgctgggca gacccatgac
     8401 cttgctgacg gtgcg
//
