LOCUS       sequence-415182-        5233 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for hlpA-mkate2 amplification associated with kanamycin 
            resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5233)
  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 5233)
  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: pJethlpAmKate2Km
FEATURES             Location/Qualifiers
     source          1..5233
                     /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             1578..2372
                     /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(3065..3071,3072..3089,3090..3095))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    3110..3131
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3251..3268)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3422..4010)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3502..3521)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4181..4972,4973..5041))
                     /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     4804..4823
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5042..5146)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5214..5232
                     /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 atcagaactt atcaaggaaa atatgcacat gaaattgtac
      721 atggaaggaa cagtaaataa tcaccacttt aaatgtacct cagaaggaga aggaaaacca
      781 tatgaaggta ctcaaaccat gcgtattaag gccgttgaag gtggaccatt gccttttgcc
      841 tttgatattc ttgctacatc ttttatgtac ggatcaaaga cttttatcaa tcatacccaa
      901 ggtatcccag atttctttaa acagtcattt cctgaaggat ttacatggga acgtgtcaca
      961 acttatgaag atggtggagt attgacagca actcaagata catctttgca agatggttgt
     1021 cttatctaca atgtaaagat ccgtggagtt aattttccaa gtaatggtcc tgttatgcag
     1081 aaaaagaccc ttggatggga agcatctacc gaaacattat atcctgccga tggtggattg
     1141 gaaggtcgtg ctgatatggc attgaaactt gtcggtggag gtcaccttat ctgtaatttg
     1201 aagaccacat accgttctaa aaagccagct aagaatctta agatgcctgg tgtttactac
     1261 gtcgatcgtc gtttagaacg tatcaaagaa gcagataagg aaacttatgt tgaacagcac
     1321 gaagtagccg tcgcacgtta ttgtgatttg cctagtaaat tgggacaccg ttaataagaa
     1381 ttctatcata attgtggttt caaaatcggc tccgtcgata ctatgttata cgccaacttt
     1441 gaaaacaact ttgaaaaagc tgttttctgg tatttaaggt tttagaatgc aaggaacagt
     1501 gaattggagt tcgtcttgtt ataattagct tcttggggta tctttaaata ctgtagaaaa
     1561 gaggaaggaa ataataaatg gctaaaatga gaatatcacc ggaattgaaa aaactgatcg
     1621 aaaaataccg ctgcgtaaaa gatacggaag gaatgtctcc tgctaaggta tataagctgg
     1681 tgggagaaaa tgaaaaccta tatttaaaaa tgacggacag ccggtataaa gggaccacct
     1741 atgctgtgga acgggaaaag gacatgatgc tatggctgga aggaaagctg cctgttccaa
     1801 aggtcctgca ctttgaacgg catgatggct ggagcaatct gctcatgagt gaggccgatg
     1861 gcgtcctttg ctcggaagag tatgaagatg aacaaagccc tgaaaagatt atcgagctgt
     1921 atgcggagtg catcaggctc tttcactcca tcgacatatc ggattgtccc tatacgaata
     1981 gcttagacag ccgcttagcc gaattggatt acttactgaa taacgatctg gccgatgtgg
     2041 attgcgaaaa ctgggaagaa gacactccat ttaaagatcc gcgcgagctg tatgattttt
     2101 taaagacgga aaagcccgaa gaggaacttg tcttttccca cggcgacctg ggagacagca
     2161 acatctttgt gaaagatggc aaagtaagtg gctttattga tcttgggaga agcggcaggg
     2221 cggacaagtg gtatgacatt gccttctgcg tccggtcgat cagggaggat atcggggaag
     2281 aacagtatgt cgagctattt tttgacttac tggggatcaa gcctgattgg gagaaaataa
     2341 aatattatat tttactggat gaattgtttt agtacctaga tttagatgtc taaaaagcaa
     2401 aaacaagatc cagaaaggat cttgtttttt ctagaactat agctagcagc ctatggagtt
     2461 gtaaactgta caacactttt tataagctct ctcaatcgaa tagctacctg ctctgcaaat
     2521 tctaatgagc actagtagga ggcatatcaa atgaacttta ataaaattga tttagacaat
     2581 tggaagagaa aagagatatt taatcattat ttgaaccaac aaacgacttt atctttctag
     2641 aagatctcct acaatattct cagctgccat ggaaaatcga tgttcttctt ttattctctc
     2701 aagattttca ggctgtatat taaaacttat attaagaact atgctaacca cctcatcagg
     2761 aaccgttgta ggtggcgtgg gttttcttgg caatcgactc tcatgaaaac tacgagctaa
     2821 atattcaata tgttcctctt gaccaacttt attctgcatt ttttttgaac gaggtttaga
     2881 gcaagcttca ggaaactgag acaggaattt tattaaaaat ttaaattttg aagaaagttc
     2941 agggttaata gcatccattt tttgctttgc aagttcctca gcattcttaa caaaagacgt
     3001 ctcttttgac atgtttaaag tttaaacctc ctgtgtgaaa ttattatccg ctcataattc
     3061 cacacattat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3121 aactcacatt aattgcgttg cgctcactgc caattgcttt ccagtcggga aacctgtcgt
     3181 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3241 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3301 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3361 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3421 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3481 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3541 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3601 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3661 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3721 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3781 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3841 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3901 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3961 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4021 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4081 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4141 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4201 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4261 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4321 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4381 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4441 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4501 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4561 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4621 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4681 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4741 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4801 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4861 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4921 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4981 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5041 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5101 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5161 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5221 gtatcacgag gcc
//
