LOCUS       sequence-418282-        5740 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BTK assembled plasmid for integrating a KanR+E2-Crimson cassette 
            into the S. alvi genome in place of the clpA gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5740)
  AUTHORS   Lariviere PJ, Zuberi Ashraf AHM, Leonard SP, Miller LG, Moran NA, 
            Barrick JE
  TITLE     Single-step genome engineering in the bee gut symbiont Snodgrassella
            alvi
  JOURNAL   bioRxiv 2023.09.19.558440
REFERENCE   2  (bases 1 to 5740)
  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: "bioRxiv 
            2023.09.19.558440"
COMMENT     Sequence Label: pBTK1061
FEATURES             Location/Qualifiers
     source          1..5740
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1110..1784)
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2009)"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHM
                     ALKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(1367..1386)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2039..2058)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2050..2865
                     /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             2050..2865
                     /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     complement(2123..2142)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3902..4490)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3982..4001)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4593..4697
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4698..4766,4767..5558)
                     /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     complement(4916..4935)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcaggcaaat caggtgactt ttctgaaaac aaccggatac gattttggta tacgcagctt
       61 tgatgatcag cttattgcac cgctgcaaaa agttttaaat cagccactgc ctgcgaatcg
      121 gtcacggctg attgcgctgc atatcatagg ttctcatcct aatccgtgcg accgtcttcc
      181 acaaccagcg catcactatg tcgataataa caacagtaac tgctatattg acactatccg
      241 gcagacagac aatttactaa gtcagatata tagtgcatta cagcacagcg gtcagccatt
      301 cagtatgttg tatttcagcg atcacggttt aagtcacgat aaaaacagtt atgaaaaaaa
      361 cagcctcgtg cgttctgaaa gtattcttcg tcataatgat cacttctatc agaactacca
      421 cgtaccatta gtgattatca gcagcaatga aaccggacaa ttacgtaaca aagcacggcg
      481 cagcggttta gaattgctgg atggagttgc acaatggctg ggaatcagca ccccgcaact
      541 gccgtatagt aaacaatttt tcagtgaaac ggacagtact cagctagagg ttctggatct
      601 caatcagcgg ttacaacctg tagataagct gcaaaacgat cctctccctg cacaattgca
      661 gtgacaatta cataaaaaaa caatctctct gtttttttac aaagcagaga gattaaattt
      721 ttcaaataat ttcagattaa agctttacat ttgatatgaa tcaaaacact tctgaacaaa
      781 aacattataa tgaatttact ttgattggtt tcctcttgat gtgtgtgttt aagtgtggct
      841 cttcgccaca ctttttttta tcttaatttt gacattctta acataattat tcaattgtta
      901 ctgattcaaa tttgttctga tcagcttcct gagaatccgc aaaatccagt actacttcat
      961 ctgactgcca gcgacgcagc tgacaaatgc tctttcccta aactcccccc ataaaaaaac
     1021 ccgccgaagc gggtttttac gttatttgcg gattaacgat tacggattta caattcgtcg
     1081 tgcttgtaca attcgtccat accgtgagtc tggaacaggt ggtggcgggc ctcggcgcgc
     1141 tcgtactgct ccaccacggt gtagtcctcg ttgtgggagg tgatgtcgag cttgtagtcc
     1201 acgtagtggt agccgggcag cttcacgggc ttcttggcca tgtagatgga cttgaactca
     1261 cacaggtagt ggccgccgcc cttcagcttc agcgccatgt ggttctcgcc cttcagcacg
     1321 ccgtcgcggg ggtagttgcg ctcagtggag ggctcccagc ccagagtctt cttctgcatt
     1381 acggggccgt cggaggggaa gttcacgccg atgaacttca cgtggtagat gagggtgccg
     1441 tcctgcaggg aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa gttcatcacg
     1501 cgctcccact tgaagccctc ggggaaggac tgcttgaggt agtcggggat gtcggcgggg
     1561 tgcttgatgt acgccttgga gccgtagaag aactgggggg acaggatgtc ccaggcgaag
     1621 ggcagggggc cgcccttggt cacttgcagc ttggcggtct gggtgccctc gtagggcttg
     1681 ccctcgccca cgccctcgat ctcgaactcg tggccgttca cggagccctc catgtgcacc
     1741 ttgaagcgca tgaagggctt gatgacgttc tcagtgctat ccatatgtaa tatctcctct
     1801 gtttctgtat aacagtacta tgtgattata ccagccccct cactacatgt caagaataaa
     1861 ctgccaaagc gttcgtctgc aattatcggc cagttcacat actgatcctt caactcagca
     1921 aaagttcgat ttattcaaca aagccacgtt gtgtctcaaa atctctgatg ttacattgca
     1981 caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     2041 aggggtgtta tgagccatat tcaacgggaa acgtcttgct ccaggccgcg attaaattcc
     2101 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     2161 gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     2221 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     2281 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     2341 accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt
     2401 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     2461 aattgtcctt ttaacagcga tcgcgtattt cgcctcgctc aggcgcaatc acgaatgaat
     2521 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     2581 gtctggaaag aaatgcataa gcttttgcca ttctcaccgg attcagtcgt cactcatggt
     2641 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     2701 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     2761 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     2821 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat
     2881 tggttgtaac accgttgctg tgccgaaatc atatccgacc tcctgacaaa gtaacgttat
     2941 caatagcgac tgcaaccaat tttattcact tgtataaaca acatgaagca tacacgataa
     3001 aattcaagta gcgcccgcag aactaatatt tcacccgtaa ttacccaaac aggcaattca
     3061 ggcctcttct accaaacact gtaaaggaaa accggctgct tccgagcgtg acaaaacctg
     3121 attacatttt gtttctgcaa tatctttttg aaacacaccg catatcccgc tgcctgattc
     3181 atgcaccatc agcataatag ctatggcctg cgtatgtggt aaatgaaaca catccattaa
     3241 aattgcaatt acaaagtcca ttgtcgtgta atcatcattc agcaaaacca ctttatagcg
     3301 ctttggcggc attatcatgg atttctggct ctggctaaca atttgtatct tcgaatttga
     3361 cattgtctat taatctacat gtatctgtgg gtttttagta acggttttaa taattttatc
     3421 atttttcggt aagcttgctt gacgttagtt cttaacctat gtaaaaagac tacttaacaa
     3481 gtcagacata gactgccaga caaactgttg tacagtcgtt cactattttt ttggcctttt
     3541 tgtgtcgtaa acttgttctt tattttgttt tttatgtata ggaagtttca tggcaactgg
     3601 tattgttaag tggtttaacg acgctaaagg ttttggtttt attaccccag atgaaggtgg
     3661 cgaagatttg tttgcacatt tctctgcaat caacatgaat ggtttcaaaa ccttgaaaga
     3721 aggtcaacgt gtatcttttg atgtgaccac cggtccaaaa ggtaaacaag cggctaatat
     3781 tcaggaattc aggggcggcc gcacggttat ccacagaatc aggggataac gcaggaaaga
     3841 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3901 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3961 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4021 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4081 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4141 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4201 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4261 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4321 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4381 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4441 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4501 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4561 tcatgacagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4621 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4681 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4741 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4801 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4861 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4921 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4981 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5041 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5101 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5161 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5221 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5281 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5341 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5401 ccggtgagcg tgggtcccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5461 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5521 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5581 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5641 tttttgataa tcgcggccgc tgtagaattc tggatactgt ctggttatct aatcaggggg
     5701 ctctcggaca atacgataca tcaatttcca ctattgctca
//
