LOCUS Exported 6748 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Encodes a Fluorescent Reporter for Hydrogen Sulfide in Mammalian Cells. ACCESSION . VERSION . KEYWORDS pcDNA3-hsGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6748) AUTHORS Chen ZJ, Ai HW TITLE A highly responsive and selective fluorescent probe for imaging physiological hydrogen sulfide. JOURNAL Biochemistry. 2014 Sep 23;53(37):5966-74. doi: 10.1021/bi500830d. Epub 2014 Sep 5. PUBMED 25141269 REFERENCE 2 (bases 1 to 6748) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6748 /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" regulatory 898..907 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 1137..1158 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1270..1291) /label=EGFP-N /note="EGFP, reverse primer" CDS 1663..1680 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" misc_feature 1696..2284 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(1749..1769) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2167..2178) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" promoter complement(2300..2318) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2301..2318) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2338..2355) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2344..2568 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2614..3042 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2701..2720) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2911..2932 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3051..3071) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3056..3386 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3237..3372 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3299..3318 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3453..4247 /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(3507..3526) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4117..4136 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4421..4542 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4458..4477) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4512..4531 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4591..4607) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4591..4607) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4604..4626) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4615..4631 /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(4639..4669) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4684..4705 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4822..4839) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4993..5581) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5073..5092) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5752..6612) /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 6375..6394 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6613..6717) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 gcttgccgcc 901 accatgggct ccagcccgta caacagccac aaggtctata tcaccgccga caagcagaag 961 aacggcatca aggtgaactt caagatccgc cacaacgtgg aggacggcag cgtgcagctc 1021 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 1081 cactacctga gcacccagtc cgtgctgagc aaagacccca acgagaagcg cgatcacatg 1141 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 1201 gtggacggcg gatccggtgg caccggagtg agcaagggcg aggagctgtt caccggggtg 1261 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgaggggc 1321 gaaggcgagg gcgatgccac caacggcaag ctgaccctga agttcatctg caccaccggc 1381 aagctgcccg tgccctggcc caccctcgtg accaccttga cctagggcgt gcagtgcttc 1441 tcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 1501 tacgtccagg agcgcaccat cttcttcaag gacgacggca cctacaagac ccgcgccgag 1561 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 1621 gaggacggca acatcctggg gcacaagctg gagtacaact ggcaccatca ccatcaccat 1681 taactcgagg tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt 1741 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat 1801 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct 1861 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct 1921 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc 1981 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg 2041 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc 2101 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac 2161 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg 2221 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc 2281 ccgctctaga gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct 2341 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 2401 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 2461 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 2521 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg 2581 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg 2641 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 2701 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 2761 taaatcgggg catcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 2821 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 2881 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 2941 tcaaccctat ctcggtctat tcttttgatt tataagggat tttggggatt tcggcctatt 3001 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg 3061 tcagttaggg tgtggaaagt ccccaggctc cccaggcagg cagaagtatg caaagcatgc 3121 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta 3181 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc 3241 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta 3301 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct 3361 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat 3421 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag 3481 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg 3541 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca 3601 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc 3661 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg 3721 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg 3781 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta 3841 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag 3901 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac 3961 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg 4021 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg 4081 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg 4141 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg 4201 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg 4261 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc 4321 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct 4381 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta 4441 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 4501 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc 4561 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 4621 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 4681 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 4741 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 4801 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 4861 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 4921 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 4981 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 5041 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 5101 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 5161 cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta 5221 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 5281 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 5341 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 5401 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 5461 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 5521 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 5581 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 5641 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 5701 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 5761 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 5821 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 5881 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 5941 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 6001 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 6061 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 6121 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 6181 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 6241 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 6301 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 6361 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 6421 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 6481 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 6541 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 6601 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 6661 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 6721 atttccccga aaagtgccac ctgacgtc //