LOCUS       sequence-296867-        7978 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 7978)
  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 7978)
  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..7978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     148..168
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    253..826
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(420..437)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     647..666
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(2287..2304)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2293..2517
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2563..2991
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2650..2669)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2860..2881
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3000..3020)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3005..3334
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3185..3320
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3247..3266
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    3574..3695
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3611..3630)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3665..3684
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3744..3760)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3744..3760)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3757..3779)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3768..3784
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3792..3798,3799..3816,3817..3822))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3837..3858
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3975..3992)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4146..4734)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4226..4245)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4905..5696,5697..5765))
                     /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     5528..5547
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5766..5870)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5945..5964)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6136..6515
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6516..6719
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6670..6690
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6764..6783
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6764..6782
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6859..7560
                     /codon_start=1
                     /product="orange-red fluorescent protein that is excitable 
                     by cyan light and suitable as an acceptor for blue-emitting
                     luciferase (Chu et al., 2016)"
                     /label=CyOFP1
                     /translation="MVSKGEELIKENMRSKLYLEGSVNGHQFKCTHEGEGKPYEGKQTN
                     RIKVVEGGPLPFAFDILATHFMYGSKVFIKYPADLPDYFKQSFPEGFTWERVMVFEDGG
                     VLTATQDTSLQDGELIYNVKVRGVNFPANGPVMQKKTLGWEPSTETMYPADGGLEGRCD
                     KALKLVGGGHLHVNFKTTYKSKKPVKMPGVHYVDRRLERIKEADNETYVEQYEHAVARY
                     SNLGGGMDELYK"
     CDS             7570..7623
                     /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             join(7630..7978,1..251)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7630..7649)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
ORIGIN
        1 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca
       61 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
      121 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
      181 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
      241 ccggtgcctg aacccctctc cctccccccc ccntaacgtt actggccgaa gccgcttgga
      301 ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa
      361 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc
      421 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc
      481 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg
      541 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
      601 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
      661 cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct
      721 ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc
      781 cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataatgtg atggttgaga
      841 tgttgccaac cgtagctgtg ctcgttttgg ctgtcagcgt agtcgctaaa gataatacaa
      901 cacttcagga gttcgcaaca tctaagggag aagagttgtt tactggcgta gtacccattc
      961 ttgtggaact tgacggcgat gttaatggtc ataagttctc tgtgtctggt gagggcgagg
     1021 gggatgctac atatggtaaa ttgaccctca aatttatttg tacaaccggg aagctccctg
     1081 taccctggcc tactctcgtg accaccctca cctacggggt acaatgtttt tccagatacc
     1141 ccgatcacat gaagcgccac gatttcttca aaagtgccat gcccgagggt tacgtccagg
     1201 aacgcacaat atttttcaag gatgatggaa attacaaaac ccgcgccgag gttaagttcg
     1261 agggggatac cctcgttaac cggattgaac tgaagggtat cgactttaag gaggacggta
     1321 atatattggg gcataaattg gaatacaact ataacgacca ccaggtgtat atcatggccg
     1381 ataaacagaa gaacggcatc aaagcaaact ttaaaattag gcataacata gaggatggtg
     1441 gcgttcagct tgctgatcac tatcaacaga atacccctat aggtgatgga cccgtgctcc
     1501 tcccagataa ccattacttg tttacaactt ctacactctc taaggacccc aacgaaaaga
     1561 gagaccacat ggtgctcttg gagttcgtaa ctgccgccgg aattacacat ggtatggacg
     1621 agctctacaa ggatatttcc gggggggtct ttacacttga agatttcgtc ggggattgga
     1681 gacaaactgc cggttataat caggatcagg tgcttgagca aggggggctg agctcccttt
     1741 ttcaaaagct gggtgtatct gtcaccccta tccaaaagat tgtgcttagc ggtgaaaatg
     1801 gtctcaaatt cgacatccac gtgatcatcc catacgaggg tcttagtgga ttccagatgg
     1861 ggctgataga gatgattttc aaagttgttt accctgtcga cgaccaccat ttcaaaatca
     1921 ttctgcacta cggaaccctc gtcatagacg gggtcacacc caacatgata gattactttg
     1981 ggcgacccta tgaagggata gcagtattcg acggcaaaaa gataaccgtg acaggcaccc
     2041 tgtggaatgg aaacaaaatt atagatgagc gcctgatcaa ccctgacggt tctttgttgt
     2101 tcagggttac aatcaatggt gtcacaggct ggaggctctg tgagaacatc ctggctcagt
     2161 tggagaatgg ggggatctcc ctcttggtgc aaaatacatc atggatgttg cttcttcttc
     2221 ttagcttgag cctcttgcag gcattggatt tcatcagtct ctaacccgtt taaacccgct
     2281 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2341 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     2401 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     2461 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     2521 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     2581 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2641 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2701 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2761 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2821 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2881 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2941 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     3001 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3061 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3121 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3181 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3241 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     3301 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     3361 tttcggatct gatcaagaga caggatgagg atcgtttcgc gcgggactct ggggttcgaa
     3421 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     3481 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     3541 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     3601 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     3661 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     3721 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3781 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3841 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3901 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3961 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4021 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4081 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4141 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4201 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4261 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4321 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4381 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4441 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4501 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4561 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4621 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4681 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4741 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4801 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4861 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4921 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4981 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5041 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5101 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5161 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5221 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5281 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5341 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5401 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5461 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5521 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5581 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5641 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5701 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5761 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5821 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5881 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     5941 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     6001 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     6061 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     6121 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     6181 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     6241 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     6301 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     6361 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     6421 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     6481 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     6541 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     6601 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     6661 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
     6721 tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg actcactata
     6781 gggagaccca agctggctag cgtttaaact taagcttggt accgagctcg gatccactag
     6841 tccagtgtgg tggaattcat ggtgagcaag ggcgaggagc tgatcaagga gaacatgaga
     6901 agcaagctgt acctggaagg cagcgtgaac ggccaccagt tcaagtgcac ccacgaaggg
     6961 gagggcaagc cctacgaggg caagcagacc aacaggatca aggtggtgga gggaggcccc
     7021 ctgccgttcg cattcgacat cctggccacc cactttatgt acgggagcaa ggtgttcatc
     7081 aagtaccccg ccgacctccc cgattatttt aagcagtcct tccctgaggg cttcacatgg
     7141 gagagagtca tggtgttcga agacgggggc gtgctgaccg ccacccagga caccagcctc
     7201 caggacggcg agctcatcta caacgtcaag gtcagagggg tgaacttccc agccaacggc
     7261 cccgtgatgc agaagaaaac actgggctgg gagcccagca ccgagaccat gtaccccgct
     7321 gacggcggcc tggaaggcag atgcgacaag gccctgaagc tcgtgggcgg gggccacctg
     7381 cacgtcaact tcaagaccac atacaagtcc aagaaacccg tgaagatgcc cggcgtccac
     7441 tacgtggacc gcagactgga aagaatcaag gaggccgaca acgagaccta cgtcgagcag
     7501 tacgagcacg ctgtggccag atactccaac ctgggcggag gcatggacga gctgtacaag
     7561 gcggccgctg agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc
     7621 ccttccggga tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     7681 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     7741 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     7801 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     7861 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     7921 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcacc
//
