LOCUS       sequence-415196-        5113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for hlpA-mneongreen amplification associated with kanamycin
            resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5113)
  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 5113)
  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: pJethlpAmNeonGreenKm
FEATURES             Location/Qualifiers
     source          1..5113
                     /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             1594..2388
                     /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(2945..2951,2952..2969,2970..2975))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    2990..3011
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3131..3148)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3302..3890)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3382..3401)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4061..4852,4853..4921))
                     /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     4684..4703
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4922..5026)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5094..5112
                     /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 atcagtctct gaagaagata atatggctag tttgcctgct
      721 acccacgaat tacacatttt tggatcaatc aatggagtcg attttgatat ggttggtcaa
      781 ggaaccggta atccaaatga tggatacgaa gaattgaatc ttaagagtac taagggtgat
      841 ttacagtttt ctccatggat tttggtccct catatcggat acggttttca ccaatacttg
      901 ccatatcctg atggaatgtc tccttttcag gctgcaatgg ttgatggatc aggttatcaa
      961 gtccatcgta caatgcagtt tgaagatggt gctagtctta ccgttaatta tcgttacaca
     1021 tatgaaggat ctcacattaa aggtgaagcc caagtaaagg gaacaggttt tccagcagat
     1081 ggacctgtta tgactaattc acttaccgcc gctgattggt gtcgtagtaa aaagacttat
     1141 ccaaatgata agacaatcat ctctactttt aagtggtcat acacaactgg aaatggtaaa
     1201 cgttatcgtt caactgctcg taccacatac acctttgcaa aaccaatggc agccaattac
     1261 cttaagaatc aacctatgta cgtctttcgt aagacagaac ttaagcattc taagactgaa
     1321 cttaatttta aggaatggca gaaggctttt actgatgtaa tgggtatgga tgaattgtat
     1381 aaataataag aattaattct atcataattg tggtttcaaa atcggctccg tcgatactat
     1441 gttatacgcc aactttgaaa acaactttga aaaagctgtt ttctggtatt taaggtttta
     1501 gaatgcaagg aacagtgaat tggagttcgt cttgttataa ttagcttctt ggggtatctt
     1561 taaatactgt agaaaagagg aaggaaataa taaatggcta aaatgagaat atcaccggaa
     1621 ttgaaaaaac tgatcgaaaa ataccgctgc gtaaaagata cggaaggaat gtctcctgct
     1681 aaggtatata agctggtggg agaaaatgaa aacctatatt taaaaatgac ggacagccgg
     1741 tataaaggga ccacctatgc tgtggaacgg gaaaaggaca tgatgctatg gctggaagga
     1801 aagctgcctg ttccaaaggt cctgcacttt gaacggcatg atggctggag caatctgctc
     1861 atgagtgagg ccgatggcgt cctttgctcg gaagagtatg aagatgaaca aagccctgaa
     1921 aagattatcg agctgtatgc ggagtgcatc aggctctttc actccatcga catatcggat
     1981 tgtccctata cgaatagctt agacagccgc ttagccgaat tggattactt actgaataac
     2041 gatctggccg atgtggattg cgaaaactgg gaagaagaca ctccatttaa agatccgcgc
     2101 gagctgtatg attttttaaa gacggaaaag cccgaagagg aacttgtctt ttcccacggc
     2161 gacctgggag acagcaacat ctttgtgaaa gatggcaaag taagtggctt tattgatctt
     2221 gggagaagcg gcagggcgga caagtggtat gacattgcct tctgcgtccg gtcgatcagg
     2281 gaggatatcg gggaagaaca gtatgtcgag ctattttttg acttactggg gatcaagcct
     2341 gattgggaga aaataaaata ttatatttta ctggatgaat tgttttagta cctaaattaa
     2401 ttctaatgag cactagtagg aggcatatca aatgaacttt aataaaattg atttagacaa
     2461 ttggaagaga aaagatatat ttaatcatta tttgaaccaa caaacgactt atctttctag
     2521 aagatctcct acaatattct cagctgccat ggaaaatcga tgttcttctt ttattctctc
     2581 aagattttca ggctgtatat taaaacttat attaagaact atgctaacca cctcatcagg
     2641 aaccgttgta ggtggcgtgg gttttcttgg caatcgactc tcatgaaaac tacgagctaa
     2701 atattcaata tgttcctctt gaccaacttt attctgcatt ttttttgaac gaggtttaga
     2761 gcaagcttca ggaaactgag acaggaattt tattaaaaat ttaaattttg aagaaagttc
     2821 agggttaata gcatccattt tttgctttgc aagttcctca gcattcttaa caaaagacgt
     2881 ctcttttgac atgtttaaag tttaaacctc ctgtgtgaaa ttattatccg ctcataattc
     2941 cacacattat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3001 aactcacatt aattgcgttg cgctcactgc caattgcttt ccagtcggga aacctgtcgt
     3061 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3121 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3181 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3241 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3301 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3361 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3421 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3481 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3541 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3601 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3661 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3721 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3781 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3841 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3901 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3961 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4021 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4081 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4141 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4201 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4261 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4321 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4381 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4441 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4501 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4561 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4621 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4681 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4741 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4801 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4861 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4921 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4981 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5041 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5101 gtatcacgag gcc
//
