LOCUS Exported 8584 bp ds-DNA circular SYN 01-MAR-2022 DEFINITION Encodes codon-optimized AF mutant of M. mazei pyrrolysine (Pyl) tRNA synthetase fused to a nuclear export signal (NESPylRSAF),tRNACUAPyl. ACCESSION . VERSION . KEYWORDS pcDNA3.1(+)_U6 tRNAPyl_CMV NESPylRS(AF)_IRES_eRF1(E55D)-HA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8584) AUTHORS Arsic A, Hagemann C, Stajkovic N, Schubert T, Nikic-Spiegel I TITLE Minimal genetically encoded tags for fluorescent protein labeling in living neurons. JOURNAL Nat Commun. 2022 Jan 14;13(1):314. doi: 10.1038/s41467-022-27956-y. PUBMED 35031604 REFERENCE 2 (bases 1 to 8584) AUTHORS . TITLE Direct Submission JOURNAL Exported Mar 1, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8584 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 7..386 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 387..590 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 541..561 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 635..654 /label=T7 /note="T7 promoter, forward primer" promoter 635..653 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 673..682 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 703..729 /codon_start=1 /product="nuclear export signal from the HIV Rev protein (Fischer et al., 1995)" /label=NES /translation="LPPLERLTL" misc_feature 2119..2670 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2263..2280) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2490..2509 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" regulatory 2695..2704 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2725..2751 /codon_start=1 /product="nuclear export signal from the HIV Rev protein (Fischer et al., 1995)" /label=NES /translation="LPPLERLTL" CDS 2755..2796 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 4105..4131 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(4182..4199) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4188..4412 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 4458..4886 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4545..4564) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4755..4776 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4895..4915) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4900..5230 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5081..5216 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5143..5162 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 5278..5325 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 5344..5718 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 5848..5969 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5885..5904) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5939..5958 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(6018..6034) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6018..6034) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6031..6053) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6042..6058 /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(6066..6096) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6111..6132 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6249..6266) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6420..7008) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6500..6519) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7179..8039) /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 7802..7821 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8040..8144) /gene="bla" /label=AmpR promoter promoter complement(8276..8516) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(8326..8345) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(8496..8516) /label=hU6-F /note="Human U6 promoter, forward primer" ORIGIN 1 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 61 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 121 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 181 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 241 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 301 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 361 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 421 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 481 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 541 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 601 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 661 aagctggcta gcgccaccat ggcttgccct gtgcctctgc agctgcctcc cctggaacgc 721 ctgaccctgg acgataagaa accactgaac accctgatta gcgccaccgg cctgtggatg 781 tccaggaccg gcacaatcca caagatcaag caccacgagg tgagcagatc caaaatctac 841 atcgagatgg cctgcggcga ccacctggtg gtgaacaaca gcaggagcag caggaccgcc 901 cgggccctga ggcaccacaa gtatcgcaag acatgcaagc ggtgtcgcgt gtccgacgag 961 gatctgaaca agttcctgac caaggccaat gaggatcaga catccgtgaa ggtgaaggtg 1021 gtgtctgccc caacccggac aaagaaggca atgccaaaga gcgtggccag ggcccccaag 1081 cctctggaga acaccgaggc agcacaggca cagccatctg gcagcaagtt tagcccagca 1141 atccccgtga gcacccagga gtccgtgtct gtgcctgcca gcgtgtccac atctatctct 1201 agcatctcta ccggagcaac agccagcgcc ctggtgaagg gcaacaccaa tcctatcaca 1261 agcatgtccg ccccagtgca ggccagcgcc cctgccctga ccaagtccca gacagaccgc 1321 ctggaggtgc tgctgaaccc taaggatgag atcagcctga acagcggcaa gccattcagg 1381 gagctggagt ccgagctgct gtctaggaga aagaaggacc tgcagcaaat ctacgccgag 1441 gagagggaga actatctggg caagctggag agggagatca ccagattctt tgtggataga 1501 ggctttctgg agatcaagtc cccaatcctg atccccctgg agtacatcga gcggatgggc 1561 atcgacaacg atacagagct gtctaagcag attttcaggg tggacaagaa tttttgcctg 1621 aggcccatgc tggccccaaa cctggccaat tacctgagga agctggaccg cgccctgccc 1681 gaccccatca agattttcga gatcggccca tgttatagaa aggagtctga tggcaaggag 1741 cacctggagg agtttaccat gctgaatttt tgccagatgg gctctggctg tacccgggag 1801 aacctggaga gcatcatcac agacttcctg aatcacctgg gcatcgactt taagatcgtg 1861 ggcgatagct gtatggtgtt tggcgacaca ctggatgtga tgcacggcga tctggagctg 1921 tcctctgccg tggtgggacc tatcccactg gacagggagt ggggcatcga taagccctgg 1981 atcggagcag gattcggcct ggagcgcctg ctgaaggtga agcacgactt taagaacatc 2041 aagcgggctg ccagaagcga aagttattac aatgggatta gcacaaacct gtgaaagctt 2101 ggtaccgagc tcggatccac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt 2161 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac 2221 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc 2281 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa 2341 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg 2401 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg 2461 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc 2521 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat 2581 gctttacatg tgtttagtcg aggttaaaaa acgtctaggc cccccgaacc acggggacgt 2641 ggttttcctt tgaaaaacac gatgataata gaattcagac ccaagctggc tagcgccacc 2701 atggcgtgcc cggtgccgct gcagctgccg ccgctggaac gcctgaccct ggatggcaag 2761 cctattccca accccctgct gggcctggat agcaccgctg acgacccctc cgctgccgat 2821 agaaacgtgg aaatctggaa gatcaagaaa ctgattaaga gcctggaggc tgccagagga 2881 aacggcacca gcatgatctc cctgattatc cctcccaagg atcagatcag cagggtcgct 2941 aagatgctcg ccgatgattt cggcaccgcc agcaatatca agagcagggt gaacagactg 3001 agcgtgctgg gcgccattac cagcgtgcag cagagactga agctgtacaa caaggtgccc 3061 cccaacggac tggtggtgta ctgtggaacc atcgtgaccg aagagggcaa ggaaaaaaaa 3121 gtgaacatcg acttcgagcc cttcaaaccc atcaacacct ccctctacct gtgcgacaat 3181 aagttccaca ccgaggccct gacagctctg ctcagcgacg actccaagtt cggcttcatt 3241 gtgatcgacg gaagcggcgc tctgttcgga accctgcagg gcaacaccag ggaggtgctc 3301 cacaagttca ccgtggacct gcctaaaaag cacggcagag gcggccagag cgctctgaga 3361 ttcgccaggc tgagaatgga gaagaggcac aactacgtga gaaaggtggc cgaaaccgcc 3421 gtgcagctgt tcattagcgg cgacaaggtg aacgtggccg gcctggtgct ggccggctcc 3481 gccgacttta agaccgagct gtcccagagc gatatgttcg atcagaggct gcaaagcaag 3541 gtgctgaagc tggtggacat cagctacgga ggcgagaacg gcttcaacca ggccatcgaa 3601 ctcagcaccg aggtgctgag caacgtgaaa ttcattcaag agaagaagct catcggcagg 3661 tacttcgacg agatcagcca ggataccggc aagtactgct ttggagtgga ggataccctg 3721 aaggccctgg agatgggcgc cgtggagatc ctgattgtgt acgagaatct ggacatcatg 3781 agatatgtgc tccactgcca gggcaccgag gaggagaaaa tcctgtacct cacccccgag 3841 caggagaagg acaaaagcca cttcaccgat aaggagaccg gccaggaaca cgagctgatt 3901 gagtccatgc ccctgctgga gtggtttgcc aacaactaca agaaattcgg cgccaccctc 3961 gagatcgtca ccgacaagtc ccaggaaggc tcccagttcg tcaagggctt cggcggcatc 4021 ggaggcatcc tcaggtacag ggtcgacttc cagggcatgg agtatcaggg cggcgacgac 4081 gagttcttcg atctggacga ctactaccca tacgatgttc cagattacgc ttaatgagcg 4141 gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag 4201 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 4261 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 4321 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 4381 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg 4441 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 4501 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 4561 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc ggggcatccc 4621 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 4681 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 4741 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 4801 ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa aaaatgagct 4861 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga 4921 aagtccccag gctccccagg caggcagaag tatgcaaagc atgcatctca attagtcagc 4981 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 5041 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc 5101 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 5161 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 5221 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt 5281 gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa 5341 accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg 5401 gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc 5461 ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg 5521 gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg 5581 gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag 5641 cagccgtggg ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg 5701 gccgaggagc aggactgaca cgtgctacga gatttcgatt ccaccgccgc cttctatgaa 5761 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat 5821 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa 5881 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 5941 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag 6001 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 6061 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 6121 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 6181 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 6241 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 6301 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 6361 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 6421 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 6481 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 6541 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 6601 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 6661 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 6721 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 6781 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 6841 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 6901 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 6961 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 7021 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 7081 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 7141 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 7201 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 7261 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 7321 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 7381 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 7441 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 7501 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 7561 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 7621 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 7681 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 7741 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 7801 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 7861 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 7921 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 7981 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 8041 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 8101 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 8161 gtgccacctg acgtcgacgg atcgggagat ctaaaaaacg gaaaccccgg gaatctaacc 8221 cggctgaacg gatttagagt ccattcgatc tacatgatca ggtttccggt gtttcgtcct 8281 ttccacaaga tatataaagc caagaaatcg aaatactttc aagttacggt aagcatatga 8341 tagtccattt taaaacataa ttttaaaact gcaaactacc caagaaatta ttactttcta 8401 cgtcacgtat tttgtactaa tatctttgtg tttacagtca aattaattcc aattatctct 8461 ctaacagcct tgtatcgtat atgcaaatat gaaggaatca tgggaaatag gccctccaat 8521 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga 8581 tata //