LOCUS       sequence-419120-        5844 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BTK assembled plasmid for integrating a KanR+E2-Crimson cassette 
            into the S. alvi genome at a neutral site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5844)
  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 5844)
  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: pBTK1054
FEATURES             Location/Qualifiers
     source          1..5844
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1752..2567)
                     /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(1752..2567)
                     /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     2475..2494
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2559..2578
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2833..3507
                     /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     3231..3250
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             complement(join(4735..5526,5527..5595))
                     /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     5358..5377
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5596..5700)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(5803..5844,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtgcgg ccgcccctga
      661 attcaacctg ccctataccg tatggttttt attcatggca ggctgtctgt ccctgtttct
      721 gtgtatttct tatatctatt accgcaacaa attcataaca acctacgcat acctacccta
      781 cggacgtgcc agcaacgccg accagcttcc caacgtcaac tggataaaat taaccagccc
      841 ctgcctgccg agacctctgt cattaatggc attgttgccg acctgcacga acctgagctc
      901 gacaaccact ggcagaaatt tcttgcccgg caaaccctca atggcatgcc tgtctacaat
      961 attcgtcagg tagaagaatt tcttaccgga cgggttaaaa ttcatcacct atatgaaaat
     1021 gatcttggtt ctttgctgcc ttcaccaacc tatgtattga ttaaacgcgt attcgacatc
     1081 gttcttatcc tgctatccgc ccctgttacc ctgccggtaa tgcttattac tgccattgcc
     1141 atcaaacttg aaagcccggg taacatcctg ttccggcaaa accgtgtagg acgcagcggc
     1201 aaagaattct gtatttataa atttcgcagc atgtgcagcg attcagagaa aaacggtgcc
     1261 cgcatggcgc aacttgggga tgcccgcgtc acccgtatcg gacacatcat ccgcaaaacc
     1321 cgcatcgacg agctgccgca gttctggaac atcctgaaag gtgacatgag cctgattggt
     1381 ccccgccctg agcaaaaagt atttgtcgac cagtttattc aggaaattcc cttttatgac
     1441 taccgccata ttgtcaaacc cggtatttcc ggctgggcgc aagttactca gggctacgct
     1501 gccgacaccg aacaaaccca gataaaaatc gaacatgatt tttactacat caaacatttt
     1561 tccttctcac tcgacatact catattcttc aaaaccctca aaaccatgct caccggcttc
     1621 ggcgcacgct aacccagcca tccccataca aaacgctacc agtaaaatct taccggtagc
     1681 gttgcaatac acttacaaaa accacatagc acaactgccg aacggtgtta caaccaatta
     1741 accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     1801 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     1861 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     1921 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     1981 tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt
     2041 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2101 attcgtgatt gcgcctgagc gaggcgaaat acgcgatcgc tgttaaaagg acaattacaa
     2161 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     2221 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     2281 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     2341 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     2401 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     2461 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     2521 tttaatcgcg gcctggagca agacgtttcc cgttgaatat ggctcataac accccttgta
     2581 ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca
     2641 atgtaacatc agagattttg agacacaacg tggctttgtt gaataaatcg aacttttgct
     2701 gagttgaagg atcagtatgt gaactggccg ataattgcag acgaacgctt tggcagttta
     2761 ttcttgacat gtagtgaggg ggctggtata atcacatagt actgttatac agaaacagag
     2821 gagatattac atatggatag cactgagaac gtcatcaagc ccttcatgcg cttcaaggtg
     2881 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgtggg cgagggcaag
     2941 ccctacgagg gcacccagac cgccaagctg caagtgacca agggcggccc cctgcccttc
     3001 gcctgggaca tcctgtcccc ccagttcttc tacggctcca aggcgtacat caagcacccc
     3061 gccgacatcc ccgactacct caagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg
     3121 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     3181 accctcatct accacgtgaa gttcatcggc gtgaacttcc cctccgacgg ccccgtaatg
     3241 cagaagaaga ctctgggctg ggagccctcc actgagcgca actacccccg cgacggcgtg
     3301 ctgaagggcg agaaccacat ggcgctgaag ctgaagggcg gcggccacta cctgtgtgag
     3361 ttcaagtcca tctacatggc caagaagccc gtgaagctgc ccggctacca ctacgtggac
     3421 tacaagctcg acatcacctc ccacaacgag gactacaccg tggtggagca gtacgagcgc
     3481 gccgaggccc gccaccacct gttccagact cacggtatgg acgaattgta caagcacgac
     3541 gaattgtaaa tccgtaatcg ttaatccgca aataacgtaa aaacccgctt cggcgggttt
     3601 ttttatgggg ggagtttagg gaaagagcat ttgtcagctg cgtcgctggc tgcaccagta
     3661 acgctgatta tctggctcta gaaaaaactc aaataccaaa atttatcaga cacggataaa
     3721 aatgattaaa cgtattgtaa atataaatac cttatcgaat aaatgcagat gactaatgtt
     3781 ctgaaaaaag ataagctaca atgaactatc acaaaccgat gcaatgctca gggagccggc
     3841 atgaccctgt ttctaccctt gatttacctg ctctgtggtc tgatcgccgg caaattaacc
     3901 cgggatacaa aacctgtttt gtctctgcta ctgacaaaat ttattatccc tgtcgtaatt
     3961 atctataata tagcgaccaa tttcagcaat atgggcgcgg tattaattac cactattgtt
     4021 gttatgagca taatgctgct tgccggtaaa tttctccgga tcaatccggt aagcaccctg
     4081 tgtttcagct atctgaatat tggctggctc ggtcttccca tagcagcctc actctttggt
     4141 aatcaggctg ccgcaatatt tatatctgcc tatatcggca gttctattgt cggtaactca
     4201 cttggggcat ggctgcttac cggcatccgg ttcaatatca gaaaactgat tcaaatgcct
     4261 cccgtcattg cccttattat tggctgctta ttgttaccac tgggtaaaca aatccgcgag
     4321 ctggatataa tttattccct cgctaaattt ctgatgagct ttcttggcat ggctattctt
     4381 ggtatgtggc tggctaaaag cagaattaca ctgaccgaca tcagaaacac cattcagacc
     4441 ttcctgaaaa aagctgcggc ttatttctgc atcctctccg ccctgcttgg ccttgcctgt
     4501 ctgtgccaac agaaccttat tctggaaaat agtgccgcac tttacttatt ttgcctgctt
     4561 cctccggcag ccaatatcat tgttcttgaa acccactatc tgggtaccgg ttactccgtt
     4621 aagaattcta cagcggccgc gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4681 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4741 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4801 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4861 tgcaatgata ccgcgggacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4921 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4981 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5041 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5101 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5161 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5221 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5281 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5341 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5401 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5461 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5521 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5581 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5641 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5701 cacatttccc cgaaaagtgc cacctgtcat gaccaaaatc ccttaacgtg agttttcgtt
     5761 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5821 gcgcgtaatc tgctgcttgc aaac
//
