LOCUS       sequence-484525-        5709 bp    DNA     circular SYN 25-AUG-2025
DEFINITION  BTK assembled plasmid for integrating a KanR+GFP 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 5709)
  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 5709)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 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: pBTK1057
FEATURES             Location/Qualifiers
     source          1..5709
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2653..2672)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2664..3479
                     /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             2664..3479
                     /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(2737..2756)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(join(4600..5391,5392..5460))
                     /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     5223..5242
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5461..5565)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(5668..5709,1..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 ctgcctgcct gagacctctg tcattaatgg cattgttgcc gacctgcacg aacctgagct
      901 cgacaaccac tggcagaaat ttcttgcccg gcaaaccctc aatggcatgc ctgtctacaa
      961 tattcgtcag gtagaagaat ttcttaccgg acgggttaaa attcatcacc tatatgaaaa
     1021 tgatcttggt tctttgctgc cttcaccaac ctatgtattg attaaacgcg tattcgacat
     1081 cgttcttatc ctgctatccg cccctgttac cctgccggta atgcttatta ctgccattgc
     1141 catcaaactt gaaagcccgg gtaacatcct gttccggcaa aaccgtgtag gacgcagcgg
     1201 caaagaattc tgtatttata aatttcgcag catgtgcagc gattcagaga aaaacggtgc
     1261 ccgcatggcg caacttgggg atgcccgcgt cacccgtatc ggacacatca tccgcaaaac
     1321 ccgcatcgac gagctgccgc agttctggaa catcctgaaa ggtgacatga gcctgattgg
     1381 tccccgccct gagcaaaaag tatttgtcga ccagtttatt caggaaattc ccttttatga
     1441 ctaccgccat attgtcaaac ccggtatttc cggctgggcg caagttactc agggctacgc
     1501 tgccgacacc gaacaaaccc agataaaaat cgaacatgat ttttactaca tcaaacattt
     1561 ttccttctca ctcgacatac tcatattctt caaaaccctc aaaaccatgc tcaccggctt
     1621 cggcgcacgc taacccagcc atccccatac aaaacgctac cagtaaaatc ttaccggtag
     1681 cgttgcaata cacttacaaa aaccacatag cacaactgcc gaacgtggca attcgtcgca
     1741 atacaacgct ttggcagttt attcttgaca tgtagtgagg gggctggtat aatcacatag
     1801 tactgttata cagaaacaga ggagatatta catatgagta aaggagaaga gcttttcaca
     1861 ggagttgtcc caatcctcgt ggaattagac ggtgatgtta atgggcacaa gttctctgtc
     1921 agtggagagg gtgaaggcga cgcaacatat ggcaagctga cccttaaatt tatttgcacc
     1981 acgggtaaac tacctgttcc atggccaaca ctggtcacta cgttcgggta tggggttcag
     2041 tgctttgcgc gctacccaga tcacatgaaa cagcacgact ttttcaagag tgcaatgccc
     2101 gaaggctatg tacaggagag aaccatcttt tttaaggatg acggcaacta taagacacgc
     2161 gccgaagtga agttcgaggg tgataccctt gttaatagaa tcgagttaaa gggtattgac
     2221 tttaaggaag atggaaatat tttaggccac aaactggaat ataactataa ctcccataat
     2281 gtgtacatta tggccgacaa gcaaaagaac ggtatcaagg ttaacttcaa gatcagacac
     2341 aacattgagg atggaagcgt tcaactagcc gaccattacc aacaaaacac cccaattggc
     2401 gatgggcctg tgctgttacc agacaaccat tacctgtcca ctcaatctgc cctttcgaaa
     2461 gatcccaacg aaaagcgcga ccacatggtc cttcttgagt ttgtcacggc tgctgggatt
     2521 acacacggca tggatgaact atacaaataa atccgtaatc gttaatccgc aaatccctct
     2581 gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
     2641 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctccaggc
     2701 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     2761 tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt
     2821 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     2881 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     2941 atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat
     3001 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     3061 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgcctc gctcaggcgc
     3121 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     3181 ggcctgttga acaagtctgg aaagaaatgc ataagctttt gccattctca ccggattcag
     3241 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     3301 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     3361 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     3421 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat
     3481 cagaattggt taattggttg taacacgcgg ctggctgcac cagtaacgct gattatctgg
     3541 ctctagaaaa aactcaaata ccaaaattta tcagacacgg ataaaaatga ttaaacgtat
     3601 tgtaaatata aataccttat cgaataaatg cagatgacta atgttctgaa aaaagataag
     3661 ctacaatgaa ctatcacaaa ccgatgcaat gctcagggag ccggcatgac cctgtttcta
     3721 cccttgattt acctgctctg tggtctgatc gccggcaaat taacccggga tacaaaacct
     3781 gttttgtctc tgctactgac aaaatttatt atccctgtcg taattatcta taatatagcg
     3841 accaatttca gcaatatggg cgcggtatta attaccacta ttgttgttat gagcataatg
     3901 ctgcttgccg gtaaatttct ccggatcaat ccggtaagca ccctgtgttt cagctatctg
     3961 aatattggct ggctcggtct tcccatagca gcctcactct ttggtaatca ggctgccgca
     4021 atatttatat ctgcctatat cggcagttct attgtcggta actcacttgg ggcatggctg
     4081 cttaccggca tccggttcaa tatcagaaaa ctgattcaaa tgcctcccgt cattgccctt
     4141 attattggct gcttattgtt accactgggt aaacaaatcc gcgagctgga tataatttat
     4201 tccctcgcta aatttctgat gagctttctt ggcatggcta ttcttggtat gtggctggct
     4261 aaaagcagaa ttacactgac cgacatcaga aacaccattc agaccttcct gaaaaaagct
     4321 gcggcttatt tctgcatcct ctccgccctg cttggccttg cctgtctgtg ccaacagaac
     4381 cttattctgg aaaatagtgc cgcactttac ttattttgcc tgcttcctcc ggcagccaat
     4441 atcattgttc ttgaaaccca ctatctgggt accggttact ccgttaagaa ttctacagcg
     4501 gccgcgatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     4561 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     4621 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4681 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     4741 ggacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     4801 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     4861 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     4921 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     4981 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5041 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5101 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5161 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5221 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5281 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     5341 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     5401 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     5461 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5521 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5581 agtgccacct gtcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5641 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5701 cttgcaaac
//
