LOCUS       sequence-422131-        5239 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for hlpA-mScarlet-I amplification associated with kanamycin
            resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5239)
  AUTHORS   Mignolet J, Staub J, van der Meer JR, Veening J-W, Libante V
  TITLE     A Modular Genetic Toolbox for Precise Gene Regulation and 
            Multi-Color Imaging in Streptococci
  JOURNAL   bioRxiv 2025.05.10.653270
REFERENCE   2  (bases 1 to 5239)
  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 
            2025.05.10.653270"
COMMENT     Sequence Label: pJethlpAmScarletKm
FEATURES             Location/Qualifiers
     source          1..5239
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     305..324
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        305..323
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1584..2378
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYAVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     promoter        complement(join(3071..3077,3078..3095,3096..3101))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    3116..3137
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3257..3274)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3428..4016)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3508..3527)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4187..4978,4979..5047))
                     /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     4810..4829
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5048..5152)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5220..5238
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gcccctgcag ccgaattata ttatttttgc caaataattt ttaacaaaag ctctgaagtc
       61 ttcttcattt aaattcttag atgatacttc atctggaaaa ttgtcccaat tagtagcatc
      121 acgctgtgag taagttctaa accatttttt tattgttgta ttatctctaa tcttactact
      181 cgatgagttt tcggtattat ctctattttt aacttggagc aggttccatt cattgttttt
      241 ttcatcatag tgaataaaat caactgcttt aacacttgtg cctgaacacc atatccatcc
      301 ggcgtaatac gactcactat agggagagcg gccgccagat cttccggatg gctcgagttt
      361 ttcagcaaga tcatggcaaa caaacaagat ttgatcgcta aagtagcaga agctacagaa
      421 ttgactaaga aagactcagc agcagcagtt gaagctgtat ttgcagcagt agctgactat
      481 cttgcagctg gtgaaaaagt tcaattgatc ggttttggta actttgaagt tcgtgagcgt
      541 gcagaacgta aaggtcgcaa cccacaaact ggtaaagaaa tgacaattgc agcttctaaa
      601 gtaccagcat tcaaagctgg taaagctctt aaagacgctg ttaaaggatc cggatctggt
      661 ggagaagctg cagctaaagg atcagtgagt aaaggcgaag ctgtaataaa ggagttcatg
      721 agatttaaag tacacatgga ggggtcaatg aacggtcatg aatttgaaat tgagggagag
      781 ggagagggaa gaccgtatga gggtacgcaa accgccaaat tgaaggtaac gaagggggga
      841 cctcttcctt tttcatggga cattcttagc ccgcagttca tgtatgggag ccgagcattt
      901 attaagcacc cagcggacat accagattat tacaaacaat ctttccctga aggtttcaag
      961 tgggagagag ttatgaactt tgaggacgga ggagctgtaa cagtgaccca agatacttca
     1021 ctagaggacg ggactcttat ttacaaagtc aaactacgag gaacgaactt tccgcctgac
     1081 ggccctgtta tgcagaaaaa gactatgggc tgggaagcct ctacggagcg cctataccca
     1141 gaggatggtg ttttgaaagg tgacattaaa atggcacttc gcctaaagga cggaggccga
     1201 tacctagcag attttaaaac aacgtataaa gctaagaaac ctgtacaaat gccaggagca
     1261 tataatgttg atcgaaaatt agatattact tctcataacg aggactacac agtggttgaa
     1321 caatatgaac gtagcgaagg tcgtcattca actgggggca tggacgaact atataaataa
     1381 taagaattct atcataattg tggtttcaaa atcggctccg tcgatactat gttatacgcc
     1441 aactttgaaa acaactttga aaaagctgtt ttctggtatt taaggtttta gaatgcaagg
     1501 aacagtgaat tggagttcgt cttgttataa ttagcttctt ggggtatctt taaatactgt
     1561 agaaaagagg aaggaaataa taaatggcta aaatgagaat atcaccggaa ttgaaaaaac
     1621 tgatcgaaaa ataccgctgc gtaaaagata cggaaggaat gtctcctgct aaggtatata
     1681 agctggtggg agaaaatgaa aacctatatt taaaaatgac ggacagccgg tataaaggga
     1741 ccacctatgc tgtggaacgg gaaaaggaca tgatgctatg gctggaagga aagctgcctg
     1801 ttccaaaggt cctgcacttt gaacggcatg atggctggag caatctgctc atgagtgagg
     1861 ccgatggcgt cctttgctcg gaagagtatg aagatgaaca aagccctgaa aagattatcg
     1921 agctgtatgc ggagtgcatc aggctctttc actccatcga catatcggat tgtccctata
     1981 cgaatagctt agacagccgc ttagccgaat tggattactt actgaataac gatctggccg
     2041 atgtggattg cgaaaactgg gaagaagaca ctccatttaa agatccgcgc gagctgtatg
     2101 attttttaaa gacggaaaag cccgaagagg aacttgtctt ttcccacggc gacctgggag
     2161 acagcaacat ctttgtgaaa gatggcaaag taagtggctt tattgatctt gggagaagcg
     2221 gcagggcgga caagtggtat gacattgcct tctgcgtccg gtcgatcagg gaggatatcg
     2281 gggaagaaca gtatgtcgag ctattttttg acttactggg gatcaagcct gattgggaga
     2341 aaataaaata ttatatttta ctggatgaat tgttttagta cctagattta gatgtctaaa
     2401 aagcaaaaac aagatccaga aaggatcttg ttttttctag aactatagct agcagcctat
     2461 ggagttgtaa actgtacaac actttttata agctctctca atcgaatagc tacctgctct
     2521 gcaaattcta atgagcacta gtaggaggca tatcaaatga actttaataa aattgattta
     2581 gacaattgga agagaaaaga gatatttaat cattatttga accaacaaac gactttatct
     2641 ttctagaaga tctcctacaa tattctcagc tgccatggaa aatcgatgtt cttcttttat
     2701 tctctcaaga ttttcaggct gtatattaaa acttatatta agaactatgc taaccacctc
     2761 atcaggaacc gttgtaggtg gcgtgggttt tcttggcaat cgactctcat gaaaactacg
     2821 agctaaatat tcaatatgtt cctcttgacc aactttattc tgcatttttt ttgaacgagg
     2881 tttagagcaa gcttcaggaa actgagacag gaattttatt aaaaatttaa attttgaaga
     2941 aagttcaggg ttaatagcat ccattttttg ctttgcaagt tcctcagcat tcttaacaaa
     3001 agacgtctct tttgacatgt ttaaagttta aacctcctgt gtgaaattat tatccgctca
     3061 taattccaca cattatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     3121 tgagctaact cacattaatt gcgttgcgct cactgccaat tgctttccag tcgggaaacc
     3181 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3241 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3301 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3361 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3421 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3481 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3541 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3601 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3661 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3721 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3781 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3841 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     3901 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3961 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4021 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4081 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4141 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4201 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4261 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4321 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4381 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4441 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4501 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4561 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4621 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4681 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4741 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4801 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4861 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4921 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4981 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5041 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5101 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5161 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     5221 ataggcgtat cacgaggcc
//
