LOCUS Exported 5722 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION precursor of the bioluminescent reporter pHLuc, for detecting extracellular pH changes in tumor acidosis. ACCESSION . VERSION . KEYWORDS SEPLuc-E2A-cyOFP1 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5722 /organism="synthetic DNA construct" /mol_type="other DNA" 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(915..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(5707..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 //