LOCUS       sequence-274012-        4563 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  precursor of the bioluminescent reporter pHluc, for detecting 
            extracellular pH changes in tumor acidosis; utilizes weaker mutant 
            of IRES and weaker precursor of Nanoluc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4563)
  AUTHORS   Ong TT, Ang Z, Verma R, Koean R, Tam JKC, Ding JL
  TITLE     pHLuc, a Ratiometric Luminescent Reporter for in vivo Monitoring of 
            Tumor Acidosis.
  JOURNAL   Front Bioeng Biotechnol. 2020 May 8;8:412. doi: 
            10.3389/fbioe.2020.00412. eCollection 2020.
  PUBMED    32457886
REFERENCE   2  (bases 1 to 4563)
  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: "Front 
            Bioeng Biotechnol."; date: "2020-05-8"; volume: "8:412. doi"; pages:
            " 10.3389/fbioe.2020.00412. eCollection 2020"
COMMENT     Sequence Label: Antares-SEPLuc C1A4E
FEATURES             Location/Qualifiers
     source          1..4563
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             685..1191
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="FTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIV
                     LSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTP
                     NMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRL
                     CERILA"
     CDS             1891..1944
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1951..2550
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHLER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1951..1970)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2447..2467
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2552..3125
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2719..2736)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2946..2965
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
ORIGIN
        1 atggtgagca agggcgagga gctgatcaag gagaacatga gaagcaagct gtacctggaa
       61 ggcagcgtga acggccacca gttcaagtgc acccacgaag gggagggcaa gccctacgag
      121 ggcaagcaga ccaacaggat caaggtggtg gagggaggcc ccctgccgtt cgcattcgac
      181 atcctggcca cccactttat gtacgggagc aaggtgttca tcaagtaccc cgccgacctc
      241 cccgattatt ttaagcagtc cttccctgag ggcttcacat gggagagagt catggtgttc
      301 gaagacgggg gcgtgctgac cgccacccag gacaccagcc tccaggacgg cgagctcatc
      361 tacaacgtca aggtcagagg ggtgaacttc ccagccaacg gccccgtgat gcagaagaaa
      421 acactgggct gggagcccag caccgagacc atgtaccccg ctgacggcgg cctggaaggc
      481 agatgcgaca aggccctgaa gctcgtgggc gggggccacc tgcacgtcaa cttcaagacc
      541 acatacaagt ccaagaaacc cgtgaagatg cccggcgtcc actacgtgga ccgcagactg
      601 gaaagaatca aggaggccga caacgagacc tacgtcgagc agtacgagca cgctgtggcc
      661 agatactcca acctgggcgg aggcttcaca ctcgaagatt tcgttgggga ctggcgacag
      721 acagccggct acaacctgga ccaagtcctt gaacagggag gtgtgtccag tttgtttcag
      781 aatctcgggg tgtccgtaac tccgatccaa aggattgtcc tgagcggtga aaatgggctg
      841 aagatcgaca tccatgtcat catcccgtat gaaggtctga gcggcgacca aatgggccag
      901 atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc atcactttaa ggtgatcctg
      961 cactatggca cactggtaat cgacggggtt acgccgaaca tgatcgacta tttcggacgg
     1021 ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca ctgtaacagg gaccctgtgg
     1081 aacggcaaca aaattatcga cgagcgcctg atcaaccccg acggctccct gctgttccga
     1141 gtaaccatca acggagtgac cggctggcgg ctgtgcgaac gcattctggc gaggcacgag
     1201 ctgatcaagg agaacatgag aagcaagctg tacctggaag gcagcgtgaa cggccaccag
     1261 ttcaagtgca cccacgaagg ggagggcaag ccctacgagg gcaagcagac caacaggatc
     1321 aaggtggtgg agggaggccc cctgccgttc gcattcgaca tcctggccac ccactttatg
     1381 tacgggagca aggtgttcat caagtacccc gccgacctcc ccgattattt taagcagtcc
     1441 ttccctgagg gcttcacatg ggagagagtc atggtgttcg aagacggggg cgtgctgacc
     1501 gccacccagg acaccagcct ccaggacggc gagctcatct acaacgtcaa ggtcagaggg
     1561 gtgaacttcc cagccaacgg ccccgtgatg cagaagaaaa cactgggctg ggagcccagc
     1621 accgagacca tgtaccccgc tgacggcggc ctggaaggca gatgcgacaa ggccctgaag
     1681 ctcgtgggcg ggggccacct gcacgtcaac ttcaagacca catacaagtc caagaaaccc
     1741 gtgaagatgc ccggcgtcca ctacgtggac cgcagactgg aaagaatcaa ggaggccgac
     1801 aacgagacct acgtcgagca gtacgagcac gctgtggcca gatactccaa cctgggcgga
     1861 ggcatggacg agctgtacaa ggcggccgct gagggcagag gaagtcttct aacatgcggt
     1921 gacgtggagg agaatcccgg cccttccggg atgaccgagt acaagcccac ggtgcgcctc
     1981 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     2041 cccgccacgc gccacaccgt cgatccggac cgccacctcg agcgggtcac cgagctgcaa
     2101 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     2161 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     2221 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     2281 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     2341 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     2401 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     2461 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     2521 tggtgcatga cccgcaagcc cggtgcctga acccctctcc ctcccccccc cctaacgtta
     2581 ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca
     2641 tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca
     2701 ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg
     2761 aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc
     2821 agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata
     2881 cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag
     2941 tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc
     3001 attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt
     3061 taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat
     3121 gataatgtga tggttgagat gttgccaacc gtagctgtgc tcgttttggc tgtcagcgta
     3181 gtcgctaaag ataatacaac acttcaggag ttcgcaacat ctaagggaga agagttgttt
     3241 actggcgtag tacccattct tgtggaactt gacggcgatg ttaatggtca taagttctct
     3301 gtgtctggtg agggcgaggg ggatgctaca tatggtaaat tgaccctcaa atttatttgt
     3361 acaaccggga agctccctgt accctggcct actctcgtga ccaccctcac ctacggggta
     3421 caatgttttt ccagataccc cgatcacatg aagcgccacg atttcttcaa aagtgccatg
     3481 cccgagggtt acgtccagga acgcacaata tttttcaagg atgatggaaa ttacaaaacc
     3541 cgcgccgagg ttaagttcga gggggatacc ctcgttaacc ggattgaact gaagggtatc
     3601 gactttaagg aggacggtaa tatattgggg cataaattgg aatacaacta taacgaccac
     3661 caggtgtata tcatggccga taaacagaag aacggcatca aagcaaactt taaaattagg
     3721 cataacatag aggatggtgg cgttcagctt gctgatcact atcaacagaa tacccctata
     3781 ggtgatggac ccgtgctcct cccagataac cattacttgt ttacaacttc tacactctct
     3841 aaggacccca acgaaaagag agaccacatg gtgctcttgg agttcgtaac tgccgccgga
     3901 attacacatg gtatggacga gctctacaag gatatttccg ggggggtctt tacacttgaa
     3961 gatttcgtcg gggattggag acaaactgcc ggttataatc aggatcaggt gcttgagcaa
     4021 ggggggctga gctccctttt tcaaaagctg ggtgtatctg tcacccctat ccaaaagatt
     4081 gtgcttagcg gtgaaaatgg tctcaaattc gacatccacg tgatcatccc atacgagggt
     4141 cttagtggat tccagatggg gctgatagag atgattttca aagttgttta ccctgtcgac
     4201 gaccaccatt tcaaaatcat tctgcactac ggaaccctcg tcatagacgg ggtcacaccc
     4261 aacatgatag attactttgg gcgaccctat gaagggatag cagtattcga cggcaaaaag
     4321 ataaccgtga caggcaccct gtggaatgga aacaaaatta tagatgagcg cctgatcaac
     4381 cctgacggtt ctttgttgtt cagggttaca atcaatggtg tcacaggctg gaggctctgt
     4441 gagaacatcc tggctcagtt ggagaatggg gggatctccc tcttggtgca aaatacatca
     4501 tggatgttgc ttcttcttct tagcttgagc ctcttgcagg cattggattt catcagtctc
     4561 taa
//
