LOCUS Exported 9295 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-IRES-Antares SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9295) 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 9295) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9295 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(23..40) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 29..253 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 299..727 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(386..405) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 596..617 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(736..756) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 741..1070 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 921..1056 /label=SV40 ori /note="SV40 origin of replication" primer_bind 983..1002 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 1137..1931 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(1191..1210) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 1801..1820 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 2105..2226 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2142..2161) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2196..2215 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(2275..2291) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2275..2291) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2288..2310) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2299..2315 /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(2323..2353) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2368..2389 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2506..2523) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2677..3265) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2757..2776) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3436..4296) /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 4059..4078 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4297..4401) /gene="bla" /label=AmpR promoter primer_bind complement(4476..4495) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 4667..5046 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 5047..5250 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 5201..5221 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 5295..5314 /label=T7 /note="T7 promoter, forward primer" promoter 5295..5313 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" misc_feature 6825..7411 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(6992..7009) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 7219..7238 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 8096..8602 /codon_start=1 /product="NanoLuc(R) luciferase" /label=Nluc /note="human codon-optimized" /translation="FTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIV LSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTP NMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRL CERILA" ORIGIN 1 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 61 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 121 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 181 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 241 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac 301 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 361 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 421 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 481 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 541 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 601 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa 661 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 721 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag 781 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc 841 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag 901 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc 961 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc 1021 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg 1081 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga 1141 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 1201 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 1261 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg 1321 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 1381 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 1441 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 1501 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 1561 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 1621 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg 1681 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 1741 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 1801 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 1861 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 1921 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc 1981 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt 2041 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca 2101 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 2161 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 2221 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata 2281 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 2341 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 2401 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 2461 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 2521 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 2581 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 2641 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 2701 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 2761 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 2821 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 2881 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 2941 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 3001 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 3061 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 3121 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 3181 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 3241 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 3301 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 3361 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 3421 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 3481 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 3541 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 3601 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 3661 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 3721 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 3781 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 3841 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 3901 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 3961 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 4021 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 4081 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 4141 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 4201 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 4261 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 4321 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 4381 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc 4441 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag 4501 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa 4561 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt 4621 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg 4681 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc 4741 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 4801 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 4861 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 4921 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 4981 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 5041 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg 5101 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 5161 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt 5221 gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac 5281 tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta gcgtttaaac 5341 ttaagcttgg taccgagctc ggatccacta gtccagtgtg gtggaattca tggttgagat 5401 gttgccaacc gtagctgtgc tcgttttggc tgtcagcgta gtcgctaaag ataatacaac 5461 acttcaggag ttcgcaacat ctaagggaga agagttgttt actggcgtag tacccattct 5521 tgtggaactt gacggcgatg ttaatggtca taagttctct gtgtctggtg agggcgaggg 5581 ggatgctaca tatggtaaat tgaccctcaa atttatttgt acaaccggga agctccctgt 5641 accctggcct actctcgtga ccaccctcac ctacggggta caatgttttt ccagataccc 5701 cgatcacatg aagcgccacg atttcttcaa aagtgccatg cccgagggtt acgtccagga 5761 acgcacaata tttttcaagg atgatggaaa ttacaaaacc cgcgccgagg ttaagttcga 5821 gggggatacc ctcgttaacc ggattgaact gaagggtatc gactttaagg aggacggtaa 5881 tatattgggg cataaattgg aatacaacta taacgaccac caggtgtata tcatggccga 5941 taaacagaag aacggcatca aagcaaactt taaaattagg cataacatag aggatggtgg 6001 cgttcagctt gctgatcact atcaacagaa tacccctata ggtgatggac ccgtgctcct 6061 cccagataac cattacttgt ttacaacttc tacactctct aaggacccca acgaaaagag 6121 agaccacatg gtgctcttgg agttcgtaac tgccgccgga attacacatg gtatggacga 6181 gctctacaag gatatttccg ggggggtctt tacacttgaa gatttcgtcg gggattggag 6241 acaaactgcc ggttataatc ttgatcaggt gcttgagcaa gggggggtga gctccctttt 6301 tcaaaacctg ggtgtatctg tcacccctat ccaaaggatt gtgcttagcg gtgaaaatgg 6361 tctcaaaatc gacatccacg tgatcatccc atacgagggt cttagtggag atcagatggg 6421 gcagatagag aagattttca aagttgttta ccctgtcgac gaccaccatt tcaaagtaat 6481 tctgcactac ggaaccctcg tcatagacgg ggtcacaccc aacatgatag attactttgg 6541 gcgaccctat gaagggatag cagtattcga cggcaaaaag ataaccgtga caggcaccct 6601 gtggaatgga aacaaaatta tagatgagcg cctgatcaac cctgacggtt ctttgttgtt 6661 cagggttaca atcaatggtg tcacaggctg gaggctctgt gagcgcatcc tggctcagtt 6721 ggagaatggg gggatctccc tcttggtgca aaatacatca tggatgttgc ttcttcttct 6781 tagcttgagc ctcttgcagg cattggattt catcagtctc taaacccctc tccctccccc 6841 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg 6901 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc 6961 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg 7021 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg 7081 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca 7141 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata 7201 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc 7261 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt 7321 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt 7381 tgaaaaacac gatgataata tggccacaac catggtgagc aagggcgagg agctgatcaa 7441 ggagaacatg agaagcaagc tgtacctgga aggcagcgtg aacggccacc agttcaagtg 7501 cacccacgaa ggggagggca agccctacga gggcaagcag accaacagga tcaaggtggt 7561 ggagggaggc cccctgccgt tcgcattcga catcctggcc acccacttta tgtacgggag 7621 caaggtgttc atcaagtacc ccgccgacct ccccgattat tttaagcagt ccttccctga 7681 gggcttcaca tgggagagag tcatggtgtt cgaagacggg ggcgtgctga ccgccaccca 7741 ggacaccagc ctccaggacg gcgagctcat ctacaacgtc aaggtcagag gggtgaactt 7801 cccagccaac ggccccgtga tgcagaagaa aacactgggc tgggagccca gcaccgagac 7861 catgtacccc gctgacggcg gcctggaagg cagatgcgac aaggccctga agctcgtggg 7921 cgggggccac ctgcacgtca acttcaagac cacatacaag tccaagaaac ccgtgaagat 7981 gcccggcgtc cactacgtgg accgcagact ggaaagaatc aaggaggccg acaacgagac 8041 ctacgtcgag cagtacgagc acgctgtggc cagatactcc aacctgggcg gaggcttcac 8101 actcgaagat ttcgttgggg actggcgaca gacagccggc tacaacctgg accaagtcct 8161 tgaacaggga ggtgtgtcca gtttgtttca gaatctcggg gtgtccgtaa ctccgatcca 8221 aaggattgtc ctgagcggtg aaaatgggct gaagatcgac atccatgtca tcatcccgta 8281 tgaaggtctg agcggcgacc aaatgggcca gatcgaaaaa atttttaagg tggtgtaccc 8341 tgtggatgat catcacttta aggtgatcct gcactatggc acactggtaa tcgacggggt 8401 tacgccgaac atgatcgact atttcggacg gccgtatgaa ggcatcgccg tgttcgacgg 8461 caaaaagatc actgtaacag ggaccctgtg gaacggcaac aaaattatcg acgagcgcct 8521 gatcaacccc gacggctccc tgctgttccg agtaaccatc aacggagtga ccggctggcg 8581 gctgtgcgaa cgcattctgg cgaggcacga gctgatcaag gagaacatga gaagcaagct 8641 gtacctggaa ggcagcgtga acggccacca gttcaagtgc acccacgaag gggagggcaa 8701 gccctacgag ggcaagcaga ccaacaggat caaggtggtg gagggaggcc ccctgccgtt 8761 cgcattcgac atcctggcca cccactttat gtacgggagc aaggtgttca tcaagtaccc 8821 cgccgacctc cccgattatt ttaagcagtc cttccctgag ggcttcacat gggagagagt 8881 catggtgttc gaagacgggg gcgtgctgac cgccacccag gacaccagcc tccaggacgg 8941 cgagctcatc tacaacgtca aggtcagagg ggtgaacttc ccagccaacg gccccgtgat 9001 gcagaagaaa acactgggct gggagcccag caccgagacc atgtaccccg ctgacggcgg 9061 cctggaaggc agatgcgaca aggccctgaa gctcgtgggc gggggccacc tgcacgtcaa 9121 cttcaagacc acatacaagt ccaagaaacc cgtgaagatg cccggcgtcc actacgtgga 9181 ccgcagactg gaaagaatca aggaggccga caacgagacc tacgtcgagc agtacgagca 9241 cgctgtggcc agatactcca acctgggcgg aggcatggac gagctgtaca agtaa //