LOCUS       sequence-415328-        5292 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for hlpA-YFP amplification associated with kanamycin 
            resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5292)
  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 5292)
  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: pJethlpAYFPKm
FEATURES             Location/Qualifiers
     source          1..5292
                     /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             1638..2432
                     /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(3124..3130,3131..3148,3149..3154))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    3169..3190
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3310..3327)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3481..4069)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3561..3580)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4240..5031,5032..5100))
                     /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     4863..4882
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5101..5205)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5273..5291
                     /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 cagctaaatc tagttctaga ggatctggtg gagaagctgc agctaaagct
      721 ggatcaaaag gagaagaact ttttacaggt gtagtaccta tcttggttga attggatggt
      781 gatgttaacg gtcacaaatt ttctgtacgt ggtgaaggtg aaggtgatgc aactaacggt
      841 aaattgacac ttaaattgat ttgtacaact ggaaaacttc ctgttccttg gcctactctt
      901 gttacaacat tgggttatgg acttatgtgt tttgctcgtt atcctgatca tatgaaacgt
      961 cacgattttt ttaaatctgc tatgccagaa ggttatgtac aagaacgtac aatttcattt
     1021 aaagatgacg gaacatataa aacacgtgct gaagtaaaat tcgaaggtga cactcttgtt
     1081 aatcgtatcg aattgaaagg aatcgatttc aaagaagatg gtaacatttt gggacacaaa
     1141 cttgaataca acttcaactc tcataatgtt tatatcacag ctgacaaaca aaaaaacggt
     1201 attaaagcta attttaaaat tcgtcacaat gttgaagatg gaggagttca attggctgat
     1261 cattatcaac aaaatacacc aatcggagac ggaccagtat tgcttccaga taaccactac
     1321 ctttcttatc aatcaaaact ttcaaaagat cctaacgaaa aacgtgacca tatggtactt
     1381 cttgaatttg ttacagcagc aggtatcact cacggtatgg acgaacttta taaataagaa
     1441 ttctatcata attgtggttt caaaatcggc tccgtcgata ctatgttata cgccaacttt
     1501 gaaaacaact ttgaaaaagc tgttttctgg tatttaaggt tttagaatgc aaggaacagt
     1561 gaattggagt tcgtcttgtt ataattagct tcttggggta tctttaaata ctgtagaaaa
     1621 gaggaaggaa ataataaatg gctaaaatga gaatatcacc ggaattgaaa aaactgatcg
     1681 aaaaataccg ctgcgtaaaa gatacggaag gaatgtctcc tgctaaggta tataagctgg
     1741 tgggagaaaa tgaaaaccta tatttaaaaa tgacggacag ccggtataaa gggaccacct
     1801 atgctgtgga acgggaaaag gacatgatgc tatggctgga aggaaagctg cctgttccaa
     1861 aggtcctgca ctttgaacgg catgatggct ggagcaatct gctcatgagt gaggccgatg
     1921 gcgtcctttg ctcggaagag tatgaagatg aacaaagccc tgaaaagatt atcgagctgt
     1981 atgcggagtg catcaggctc tttcactcca tcgacatatc ggattgtccc tatacgaata
     2041 gcttagacag ccgcttagcc gaattggatt acttactgaa taacgatctg gccgatgtgg
     2101 attgcgaaaa ctgggaagaa gacactccat ttaaagatcc gcgcgagctg tatgattttt
     2161 taaagacgga aaagcccgaa gaggaacttg tcttttccca cggcgacctg ggagacagca
     2221 acatctttgt gaaagatggc aaagtaagtg gctttattga tcttgggaga agcggcaggg
     2281 cggacaagtg gtatgacatt gccttctgcg tccggtcgat cagggaggat atcggggaag
     2341 aacagtatgt cgagctattt tttgacttac tggggatcaa gcctgattgg gagaaaataa
     2401 aatattatat tttactggat gaattgtttt agtacctaga tttagatgtc taaaaagcaa
     2461 aaacaagatc cagaaaggat cttgtttttt ctagaactat agctagcagc ctatggagtt
     2521 gtaaactgta caacactttt tataagctct ctcaatcgaa tagctacctg ctctgcaaat
     2581 tctaatgagc actataggag gcatatcaaa tgaactttaa taaaattgat ttagacaatt
     2641 ggaagagaaa agagatattt aatcattatt tgaaccaaca aacgacttta tctttctaga
     2701 agatctccta caatattctc agctgccatg gaaaatcgat gttcttcttt tattctctca
     2761 agattttcag gctgtatatt aaaacttata ttaagaacta tgctaaccac ctcatcagga
     2821 accgttgtag gtggcgtggg ttttcttggc aatcgactct catgaaaact acgagctaaa
     2881 tattcaatat gttcctcttg accaacttta ttctgcattt tttttgaacg aggtttagag
     2941 caagcttcag gaaactgaga caggaatttt attaaaaatt taaattttga agaaagttca
     3001 gggttaatag catccatttt ttgctttgca agttcctcag cattcttaac aaaagacgtc
     3061 tcttttgaca tgtttaaagt ttaaacctcc tgtgtgaaat tattatccgc tcataattcc
     3121 acacattata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3181 actcacatta attgcgttgc gctcactgcc aattgctttc cagtcgggaa acctgtcgtg
     3241 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3301 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3361 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3421 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3481 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3541 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3601 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3661 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3721 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3781 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3841 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3901 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     3961 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4021 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4081 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4141 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     4201 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     4261 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4321 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     4381 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     4441 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     4501 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     4561 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     4621 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     4681 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     4741 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     4801 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     4861 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     4921 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4981 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     5041 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     5101 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5161 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5221 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     5281 tatcacgagg cc
//
