LOCUS       sequence-415318-        4843 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for hlpA-mTurquoise2 amplification associated with 
            spectinomycin resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4843)
  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 4843)
  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: pJethlpAmTurquoise2Spc
FEATURES             Location/Qualifiers
     source          1..4843
                     /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             join(685..687,688..1401)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(685..687,688..1401)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2010)"
                     /label=mTurquoise
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(727..748)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(988..1007)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1335..1356
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(join(2675..2681,2682..2699,2700..2705))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    2720..2741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2861..2878)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3032..3620)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3112..3131)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3791..4582,4583..4651))
                     /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     4414..4433
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4652..4756)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4824..4842
                     /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 ttgtagcagt agctgactat
      481 cttgcagctg gtgaaaaagt tcaattgatc ggttttggta actttgaagt tcgtgagcgt
      541 gcagaacgta aaggtcgcaa cccacaaact ggtaaagaaa tgacaattgc agcttctaaa
      601 gtaccagcat tcaaagctgg taaagctctt aaagacgctg ttaaaggatc cggatctggt
      661 ggagaagctg aagctaaagg atcagtgagc aagggcgagg agctgttcac cggggtggtg
      721 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      781 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
      841 ctgcccgtgc cctggcccac cctcgtgacc accctgtcct ggggcgtgca gtgcttcgcc
      901 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
      961 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     1021 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     1081 gacggcaaca tcctggggca caagctggag tacaactact tcagcgacaa cgtctatatc
     1141 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag
     1201 gacggcggcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     1261 gtgctgctgc ccgacaacca ctacctgagc acccagtcca agctgagcaa agaccccaac
     1321 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1381 atggacgagc tgtacaagta ataagaattc cgaagttatc gtaacgtgac tggcaagaga
     1441 tatttttaaa acaatgaata ggtttacact tactttagtt ttatggaaat gaaagatcat
     1501 atcatatata atctagaata aaattaacta aaataattat tatctagata aaaaatttag
     1561 aagccaatga aatctataaa taaactaaat taagtttatt taattaacaa ctatggatat
     1621 aaaataggta ctaatcaaaa tagtgaggag gatatatttg aatacatacg aacaaattaa
     1681 taaagtgaaa aaaatacttc ggaaacattt aaaaaataac cttattggta cttacatgtt
     1741 tggatcagga gttgagagtg gactaaaacc aaatagtgat cttgactttt tagtcgtcgt
     1801 atctgaacca ttgacagatc aaagtaaaga aatacttata caaaaaatta gacctatttc
     1861 aaaaaaaata ggagataaaa gcaacttacg atatattgaa ttaacaatta ttattcagca
     1921 agaaatggta ccgtggaatc atcctcccaa acaagaattt atttatggag aatggttaca
     1981 agagctttat gaacaaggat acattcctca gaaggaatta aattcagatt taaccataat
     2041 gctttaccaa gcaaaacgaa aaaataaaag aatatacgga aattatgact tagaggaatt
     2101 actacctgat attccatttt ctgatgtgag aagagccatt atggattcgt cagaggaatt
     2161 aatagataat tatcaggatg atgaaaccaa ctctatatta actttatgcc gtatgatttt
     2221 aactatggac acgggtaaaa tcataccaaa agatattgcg ggaaatgcag tggctgaatc
     2281 ttctccatta gaacataggg agagaatttt gttagcagtt cgtagttatc ttggagagaa
     2341 tattgaatgg actaatgaaa atgtaaattt aactataaac tatttaaata acagattaaa
     2401 aaaattataa aaaaattgaa aaaatggtgg aaacactttt ttaatttttt tgttttatta
     2461 tttaatattt gggaaatatt cattctaatt ggaagcttca ggaaactgag acaggaattt
     2521 tattaaaaat ttaaattttg aagaaagttc agggttaata gcatccattt tttgctttgc
     2581 aagttcctca gcattcttaa caaaagacgt ctcttttgac atgtttaaag tttaaacctc
     2641 ctgtgtgaaa ttattatccg ctcataattc cacacattat acgagccgga agcataaagt
     2701 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     2761 caattgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     2821 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2881 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2941 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3001 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3061 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3121 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3181 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3241 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3301 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3361 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3421 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     3481 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3541 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3601 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3661 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3721 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3781 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3841 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3901 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3961 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4021 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4081 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4141 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4201 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4261 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4321 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4381 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4441 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4501 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4561 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4621 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4681 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4741 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     4801 ttatcatgac attaacctat aaaaataggc gtatcacgag gcc
//
