LOCUS Exported 9250 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Fluorescent FPX sensor for cAMP . ACCESSION . VERSION . KEYWORDS pPHT-PKA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9250) AUTHORS Ding Y, Li J, Enterina JR, Shen Y, Zhang I, Tewson PH, Mo GC, Zhang J, Quinn AM, Hughes TE, Maysinger D, Alford SC, Zhang Y, Campbell RE TITLE Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange. JOURNAL Nat Methods. 2015 Jan 26. doi: 10.1038/nmeth.3261. PUBMED 25622108 REFERENCE 2 (bases 1 to 9250) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9250 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 2657..2710 /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" primer_bind complement(4675..4692) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4681..4905 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 4951..5379 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5038..5057) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5248..5269 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5388..5408) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 5393..5722 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5573..5708 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5635..5654 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 5771..6796 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" polyA_signal 6926..7047 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6963..6982) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 7017..7036 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(7096..7112) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7096..7112) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(7109..7131) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 7120..7136 /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(7144..7174) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7189..7210 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(7327..7344) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7498..8083) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7578..7597) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(8254..9114) /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 8877..8896 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(9115..9219) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 901 gtttaccatg gtgagcaaga gcgaggaggt catcaaagag ttcatgcgct tcaaggtgcg 961 cttggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc 1021 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc 1081 ctgggacatc ctgtcccccc agatcatgta cggctccaag gcgtacgtga agcaccccgc 1141 cgacgtcccc gattacatga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat 1201 gcacttcgag gacggcggtc tggtgaccgt gactcaggac acctccctgc aggacggcac 1261 gctgatctac aaggtgaaga tgcgcggcac caacttcccc cccgacggcc ccgtaatgca 1321 gaagaagacc ttgggctggg attatgccac cgagcgcctg taccccgaag acggcgtgct 1381 gaagggcgag cttctggggc gcctgaagct gaaggacggc ggcctcaacc tggtggagtt 1441 caagaccatc tacatggcca agaagcccgt gcaactgccc ggctactact tcgtggacac 1501 caagctgggc atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgctc 1561 cgagggccgc caccacctgg gcatggacga gctgtacaag ggtggaggca acgccgccgc 1621 cgccaagaag ggcagcgagc aggagagcgt gaaagagttc ctagccaaag ccaaggaaga 1681 tttcctgaaa aaatgggaga ccccttctca gaatacagcc cagttggatc agtttgatag 1741 aatcaagacc cttggcaccg gctcctttgg gcgagtgatg ctggtgaagc acaaggagag 1801 tgggaaccac tacgccatga agatcttaga caagcagaag gtggtgaagc taaagcagat 1861 cgagcacact ctgaatgaga agcgcatcct gcaggccgtc aacttcccgt tcctggtcaa 1921 acttgaattc tccttcaagg acaactcaaa cctgtacatg gtcatggagt atgtagctgg 1981 tggcgagatg ttctcccacc tacggcggat tggaaggttc agcgagcccc atgcccgttt 2041 ctacgcggcg cagatcgtcc tgacctttga gtatctgcac tccctggacc tcatctaccg 2101 ggacctgaag cccgagaatc ttctcatcga ccagcagggc tatattcagg tgacagactt 2161 cggttttgcc aagcgtgtga aaggccgtac ttggaccttg tgtgggaccc ctgagtactt 2221 ggcccccgag attatcctga gcaaaggcta caacaaggct gtggactggt gggctctcgg 2281 agtcctcatc tacgagatgg ctgctggtta cccacccttc ttcgctgacc agcctatcca 2341 gatctatgag aaaatcgtct ctgggaaggt gcggttccca tcccacttca gctctgactt 2401 gaaggacctg ctgcggaacc ttctgcaggt ggatctcacc aagcgctttg ggaacctcaa 2461 gaacggggtc aatgacatca agaaccacaa gtggtttgcc acgactgact ggattgccat 2521 ctatcagaga aaggtggaag ctcccttcat accaaagttt aaaggccctg gggacacgag 2581 taactttgac gactatgagg aggaagagat ccgggtctcc atcaatgaga agtgtggcaa 2641 ggagtttact gagtttgagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa 2701 tcccgggcct agcatcgaga tcccggcggg actgacggag ctgctgcagg gcttcacggt 2761 ggaggtgctg aggcaccagc ccgcggacct gctggagttc gctctgcagc acttcacccg 2821 cctgcagcag gagaacgagc gcaaaggcac cgcgcgcttc tgccatgagg gcaggacctg 2881 gggggacctg ggcgccgctg ccgggggcgg cacccccagc aagggggtca acttcgccga 2941 ggagcccatg cagtccgact ccgaggacgg ggaggaggag gaggcggcgc ccgcggacgc 3001 aggggcgttc aatgctccag taataaaccg attcacaagg cgtgcctcag tatgtgcaga 3061 agcttataat cctgatgaag aagaagatga tgcagagtcc aggattatac atccaaaaac 3121 tgatgatcaa agaaataggt tgcaagaggc ttgcaaagac atcctgctgt ttaagaatct 3181 ggatccggag cagatgtctc aagtattaga tgccatgttt gaaaaattgg tcaaagatgg 3241 ggagcatgta attgatcaag gtgacgatgg tgacaacttt tatgtaattg atagaggcac 3301 atttgatatt tatgtgaaat gtgatggtgt tggaagatgt gttggtaact atgataatcg 3361 tgggagtttc ggcgaactgg ccttaatgta caatacaccc agagcagcta caatcactgc 3421 tacctctcct ggtgctctgt ggggtttgga cagggtaacc ttcaggagaa taattgtgaa 3481 aaacaatgcc aaaaagagaa aaatgtatga aagctttatt gagtcactgc cattccttaa 3541 atctttggag ttttctgaac gcctgaaagt agtagatgtg ataggcacca aagtatacaa 3601 cgatggagaa caaatcattg ctcagggaga ttcggctgat tcttttttca ttgtagaatc 3661 tggagaagtg aaaattacta tgaaaagaaa gggtaaatca gaagtggaag agaatggtgc 3721 agtagaaatc gctcgatgct cgcggggaca gtactttgga gagcttgccc tggtaactaa 3781 caaacctcga gcagcttctg cccacgccat tgggactgtc aaatgtttag caatggatgt 3841 gcaagcattt gaaaggcttc tgggaccttg catggaaatt atgaaaagga acatcgctac 3901 ctatgaagaa cagttagttg ccctgtttgg aacgaacatg gatattgttg aacccactgc 3961 aggtggaggc ggtggagtga gcaagggcga ggagaccatc aaagagttca tgcgcttcaa 4021 ggtgcgcatg gagggctcca tgaacggcca cgagttcgag atcgagggcg agggcgaggg 4081 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc 4141 cttcgcctgg gacatcctgt ccccccagtt catgtacggc tccgaggcgt acgtgaggca 4201 ccccgccgac atccccgatt acaagaagct gcccttcccc gagggcttca agtgggagcg 4261 cgtgatgaac ttcgaagacg gcggtctggt gaccgttacc caggactcct ccctgcagga 4321 cggcacgctg atctgcaagg tgaagatgcg cggcaccaac ttcccccccg acggccccgt 4381 aatgcagaag aagaccatgg gctgggaggc ctccaccgag atgctgtacc ccgaagacgg 4441 cgtgctgaag ggccatagct atcaggccct gaagctgaag gacggcggcc actacctggt 4501 ggagttcgag accatctaca tggccaagaa gcccgtgcaa ctgcccggcg attactgtgt 4561 ggacaccaag ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacgg 4621 gcgctccgag ggccgccacc gtctgggcat ggacgagcgg tacaagtagc gcgccctcga 4681 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 4741 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 4801 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 4861 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 4921 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg 4981 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 5041 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 5101 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 5161 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 5221 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 5281 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 5341 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca 5401 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 5461 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 5521 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc 5581 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta 5641 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt 5701 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga 5761 tcagcacgtg atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga 5821 aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt 5881 cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt 5941 ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt 6001 gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg 6061 tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga 6121 ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg 6181 accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc 6241 ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc 6301 tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc 6361 ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg 6421 gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc 6481 gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc 6541 aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag 6601 cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt 6661 ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg 6721 gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc 6781 gagggcaaag gaatagcacg tgctacgaga tttcgattcc accgccgcct tctatgaaag 6841 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct 6901 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata 6961 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 7021 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag 7081 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 7141 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 7201 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 7261 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 7321 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 7381 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 7441 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 7501 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 7561 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 7621 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 7681 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 7741 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 7801 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 7861 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 7921 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt 7981 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtttttt 8041 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 8101 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 8161 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 8221 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 8281 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 8341 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 8401 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 8461 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 8521 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 8581 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 8641 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 8701 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 8761 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 8821 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 8881 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 8941 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 9001 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 9061 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 9121 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 9181 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 9241 acctgacgtc //