LOCUS       sequence-299922-        5722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  precursor of the bioluminescent reporter pHLuc, for detecting 
            extracellular pH changes in tumor acidosis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5722)
  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 5722)
  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: SEPLuc-E2A-cyOFP1
FEATURES             Location/Qualifiers
     source          1..5722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(156..744)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(236..255)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(915..1706,1707..1775))
                     /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     1538..1557
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1776..1880)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1955..1974)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2146..2525
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2526..2729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2680..2700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2774..2793
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2774..2792
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4309..4368
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     CDS             4368..4877
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     protein_bind    5569..5590
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(join(5707..5722,1..2))
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
       61 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
      121 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
      181 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
      241 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
      301 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
      361 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
      421 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
      481 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
      541 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
      601 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
      661 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
      721 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
      781 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
      841 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
      901 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
      961 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     1021 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     1081 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     1141 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     1201 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     1261 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     1321 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     1381 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     1441 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     1501 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     1561 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     1621 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     1681 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     1741 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     1801 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     1861 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     1921 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     1981 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     2041 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     2101 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     2161 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     2221 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     2281 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     2341 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     2401 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     2461 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     2521 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     2581 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     2641 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     2701 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
     2761 ggcttatcga aattaatacg actcactata gggagaccca agctggctag cgtttaaact
     2821 taagcttggt accgagctcg gatccactag tccagtgtgg tggaattcat ggttgagatg
     2881 ttgccaaccg tagctgtgct cgttttggct gtcagcgtag tcgctaaaga taatacaaca
     2941 cttcaggagt tcgcaacatc taagggagaa gagttgttta ctggcgtagt acccattctt
     3001 gtggaacttg acggcgatgt taatggtcat aagttctctg tgtctggtga gggcgagggg
     3061 gatgctacat atggtaaatt gaccctcaaa tttatttgta caaccgggaa gctccctgta
     3121 ccctggccta ctctcgtgac caccctcacc tacggggtac aatgtttttc cagatacccc
     3181 gatcacatga agcgccacga tttcttcaaa agtgccatgc ccgagggtta cgtccaggaa
     3241 cgcacaatat ttttcaagga tgatggaaat tacaaaaccc gcgccgaggt taagttcgag
     3301 ggggataccc tcgttaaccg gattgaactg aagggtatcg actttaagga ggacggtaat
     3361 atattggggc ataaattgga atacaactat aacgaccacc aggtgtatat catggccgat
     3421 aaacagaaga acggcatcaa agcaaacttt aaaattaggc ataacataga ggatggtggc
     3481 gttcagcttg ctgatcacta tcaacagaat acccctatag gtgatggacc cgtgctcctc
     3541 ccagataacc attacttgtt tacaacttct acactctcta aggaccccaa cgaaaagaga
     3601 gaccacatgg tgctcttgga gttcgtaact gccgccggaa ttacacatgg tatggacgag
     3661 ctctacaagg atatttccgg gggggtcttt acacttgaag atttcgtcgg ggattggaga
     3721 caaactgccg gttataatct tgatcaggtg cttgagcaag ggggggtgag ctcccttttt
     3781 caaaacctgg gtgtatctgt cacccctatc caaaggattg tgcttagcgg tgaaaatggt
     3841 ctcaaaatcg acatccacgt gatcatccca tacgagggtc ttagtggaga tcagatgggg
     3901 cagatagaga agattttcaa agttgtttac cctgtcgacg accaccattt caaagtaatt
     3961 ctgcactacg gaaccctcgt catagacggg gtcacaccca acatgataga ttactttggg
     4021 cgaccctatg aagggatagc agtattcgac ggcaaaaaga taaccgtgac aggcaccctg
     4081 tggaatggaa acaaaattat agatgagcgc ctgatcaacc ctgacggttc tttgttgttc
     4141 agggttacaa tcaatggtgt cacaggctgg aggctctgtg agcgcatcct ggctcagttg
     4201 gagaatgggg ggatctccct cttggtgcaa aatacatcat ggatgttgct tcttcttctt
     4261 agcttgagcc tcttgcaggc attggatttc atcagtctcg gaagcggaca atgtactaac
     4321 tacgctttgt tgaaactcgc tggcgatgtt gaaagtaacc ccggtccgtc ttcacactcg
     4381 aagatttcgt tggggactgg cgacagacag ccggctacaa cctggaccaa gtccttgaac
     4441 agggaggtgt gtccagtttg tttcagaatc tcggggtgtc cgtaactccg atccaaagga
     4501 ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag
     4561 gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg taccctgtgg
     4621 atgatcatca ctttaaggtg atcctgcact atggcacact ggtaatcgac ggggttacgc
     4681 cgaacatgat cgactatttc ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa
     4741 agatcactgt aacagggacc ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca
     4801 accccgacgg ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt
     4861 gcgaacgcat tctggcgagg cacgaactta tcaaggagaa catgagaagc aagctgtatc
     4921 tcgaaggctc tgtaaatggg catcaattta agtgtacaca tgagggagag ggaaaaccat
     4981 acgaaggtaa acagacaaat aggattaaag tagttgaggg gggacctctc cccttcgcct
     5041 tcgacattct tgctacccat tttatgtacg ggtcaaaggt tttcattaaa taccctgctg
     5101 acctgccaga ttattttaag caaagcttcc cagagggttt tacatgggag agagttatgg
     5161 tatttgaaga tggcggcgtc cttaccgcca cccaggacac ctcccttcag gatggtgaac
     5221 tgatatataa tgtgaaggta cgcggcgtaa actttccagc taatggccca gtcatgcaaa
     5281 agaagacact gggctgggag ccatccacag agacaatgta tccagccgac ggtggattgg
     5341 aggggcgatg cgataaggct ctcaaactgg tgggtggtgg ccatttgcac gttaatttca
     5401 agaccacata caaaagtaaa aaaccagtca agatgccagg cgttcactac gtggaccgca
     5461 ggttggagag gatcaaggaa gcagacaacg aaacatacgt agagcaatac gagcatgccg
     5521 tagctcgcta caggccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5581 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5641 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5701 cgcttcctcg ctcactgact cg
//
