LOCUS Exported 9077 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION plasmid with 4xhybrid PylT cassette (Mx1201 G1 PylT hybrid mutant A41AA C55A) and G1 PylRS Y125A for amber suppression in transient or stable, piggybac-mediated, integration. ACCESSION . VERSION . KEYWORDS pAS_4xhybPylT A41AA C55A FLAG-G1 PylRS Y125A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9077) AUTHORS Meineke B, Heimgartner J, Eirich J, Landreh M, Elsasser SJ TITLE Site-Specific Incorporation of Two ncAAs for Two-Color Bioorthogonal Labeling and Crosslinking of Proteins on Live Mammalian Cells. JOURNAL Cell Rep. 2020 Jun 23;31(12):107811. doi: 10.1016/j.celrep.2020.107811. PUBMED 32579937 REFERENCE 2 (bases 1 to 9077) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9077 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 127..149 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 141..158 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 142..158 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" repeat_region 288..350 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" promoter 2164..2375 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" LTR 2388..2656 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1" regulatory 2688..2697 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2697..2720 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" misc_feature 3564..4136 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(3731..3748) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 3958..3977 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 4145..4939 /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(4199..4218) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4809..4828 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4999..5019) /label=WPRE-R /note="WPRE, reverse primer" polyA_signal 5061..5182 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5098..5117) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5152..5171 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" repeat_region 5945..5979 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(6470..6486) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6470..6486) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6483..6505) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6494..6510 /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(6518..6548) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6563..6584 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6701..6718) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6872..7460) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6952..6971) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7631..8491) /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 8254..8273 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8492..8596) /gene="bla" /label=AmpR promoter rep_origin complement(8622..9077) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(8759..8780) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 8971..8990 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc 61 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag 121 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcctcg ttcattcacg 181 tttttgaacc cgtggaggac gggcagactc gcggtgcaaa tgtgttttac agcgtgatgg 241 agcagatgaa gatgctcgac acgctgcaga acacgcagct agattaaccc tagaaagata 301 atcatattgt gacgtacgtt aaagataatc atgtgtaaaa ttgacgcatg tgttttatcg 361 gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat tatatttaca 421 cttacatact aataataaat tcaacaaaca atttatttat gtttatttat ttattaaaaa 481 aaacaaaaac tcaaaatttc ttctataaag taacaaaact tttatgaggg acagcccccc 541 cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc agcgagccgc 601 ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga 661 cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt ctcgctgctc 721 tttgagcctg cagacacctg gggggatacg gggaaaaggc ctccacggcc aactagtgag 781 acgaaaaacc gcacttgtcc gagagaccgg ggtgtcgaac cccgctttac aggttttaga 841 gacccgtttg ctcgccggag cgtcccccga ggtacccagg cggcgcacaa gctatataaa 901 cctgaaggaa atctcaactt tacacttagg tcaagttact tatcgtacta gagcttcagc 961 aggaaattta actaaaatct aatttaacca gcatagcaaa tatcatttat tcccaaaatg 1021 ctaaagtttg agataaacgg acttgatttc cggctgtttt gacactatcc agaatgcctt 1081 gcagatgggt ggggcatgct aaatactgca ggcaaaaaaa ccgcacttgt ccgagagacc 1141 ggggtgtcga accccgcttt acaggtttta gagacccgtt tgctcgccgg agcgtccccc 1201 gaggtaccca ggcggcgcac aagctatata aacctgaagg aaatctcaac tttacactta 1261 ggtcaagtta cttatcgtac tagagcttca gcaggaaatt taactaaaat ctaatttaac 1321 cagcatagca aatatcattt attcccaaaa tgctaaagtt tgagataaac ggacttgatt 1381 tccggctgtt ttgacactat ccagaatgcc ttgcagatgg gtggggcatg ctaaatactg 1441 cagcaagaaa aaccgcactt gtccgagaga ccggggtgtc gaaccccgct ttacaggttt 1501 tagagacccg tttgctcgcc ggagcgtccc ccgaggtacc caggcggcgc acaagctata 1561 taaacctgaa ggaaatctca actttacact taggtcaagt tacttatcgt actagagctt 1621 cagcaggaaa tttaactaaa atctaattta accagcatag caaatatcat ttattcccaa 1681 aatgctaaag tttgagataa acggacttga tttccggctg ttttgacact atccagaatg 1741 ccttgcagat gggtggggca tgctaaatac tgcagcgata aaaaccgcac ttgtccgaga 1801 gaccggggtg tcgaaccccg ctttacaggt tttagagacc cgtttgctcg ccggagcgtc 1861 ccccgaggta cccaggcggc gcacaagcta tataaacctg aaggaaatct caactttaca 1921 cttaggtcaa gttacttatc gtactagagc ttcagcagga aatttaacta aaatctaatt 1981 taaccagcat agcaaatatc atttattccc aaaatgctaa agtttgagat aaacggactt 2041 gatttccggc tgttttgaca ctatccagaa tgccttgcag atgggtgggg catgctaaat 2101 actgcagctc caaggccact agtgctctag aaaggatctg cgatcgctcc ggtgcccgtc 2161 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt 2221 gaacgggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc 2281 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg 2341 ttctttttcg caacgggttt gccgccagaa cacagctgaa gcttcgaggg gctcgcatct 2401 ctccttcacg cgcccgccgc cctacctgag gccgccatcc acgccggttg agtcgcgttc 2461 tgccgcctcc cgcctgtggt gcctcctgaa ctgcgtccgc cgtctaggta agtttaaagc 2521 tcaggtcgag accgggcctt tgtccggcgc tcccttggag cctacctaga ctcagccggc 2581 tctccacgct ttgcctgacc ctgcttgctc aactctacgt ctttgtttcg ttttctgttc 2641 tgcgccgtta cagatccaag ctgtgaccgg cgcctactct agagctagcc accatggact 2701 acaaggacga cgacgacaag gtggtcaagt tcaccgacag ccagatccag cacctgatgg 2761 aatacggcga caacgattgg agcgaggccg agttcgaaga tgccgccgct agagacaaag 2821 agttcagcag ccagttcagc aagctgaagt ccgccaacga caagggcctg aaggacgtga 2881 tcgccaatcc tcggaacgac ctgaccgacc tggaaaacaa gatcagagag aagctggccg 2941 ccagaggctt catcgaggtg cacaccccta tcttcgtgtc caagtctgcc ctggccaaga 3001 tgaccatcac cgaggatcac cctctgttca aacaggtgtt ctggatcgac gacaagcggg 3061 ccctgaggcc tatgcacgcc atgaacctgg ccaaagtgat gagagagctg cgggaccaca 3121 caaagggccc cgtgaagatc tttgagatcg gcagctgctt ccggaaagag agcaagagca 3181 gcacccacct ggaagagttc accatgctga acctggtgga aatgggcccc gacggcgatc 3241 ctatggaaca cctgaagatg tatatcggcg acatcatgga cgccgtgggc gtcgagtaca 3301 ccaccagcag agaagagtcc gacgtgtacg tggaaaccct ggacgtggaa atcaacggca 3361 ccgaagtggc tagcggagcc gtgggacctc acaaactgga tcctgctcac gacgtgcacg 3421 aaccttgggc cggaatcggc tttggcctgg aaagactgct gatgctgaag aacggcaaga 3481 gcaacgccag aaagaccggc aagtccatca cctacctgaa cggctacaag ctggactgat 3541 gaatcgatgg atccgcggcc gcgcccctct ccctcccccc cccctaacgt tactggccga 3601 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg 3661 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg 3721 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt 3781 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac 3841 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca 3901 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg 3961 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg 4021 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa 4081 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg ataatacctc 4141 cggaatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 4201 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 4261 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 4321 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 4381 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 4441 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc 4501 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 4561 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga 4621 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc 4681 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 4741 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 4801 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 4861 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 4921 tcttgacgag ttcttctgag tcgacaatca acctctggat tacaaaattt gtgaaagatt 4981 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc 5041 tttgtatcat gcgttaacta aacttgttta ttgcagctta taatggttac aaataaagca 5101 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 5161 ccaaactcat caatgtatct tatcatgtct ggaattgact caaatgatgt caattagtct 5221 atcagaagct catctggtct cccttccggg ggacaagaca tccctgttta atatttaaac 5281 agcagtgttc ccaaactggg ttcttatatc ccttgctctg gtcaaccagg ttgcagggtt 5341 tcctgtcctc acaggaacga agtccctaaa gaaacagtgg cagccaggtt tagccccgga 5401 attgactgga ttcctttttt agggcccatt ggtatggctt tttccccgta tccccccagg 5461 tgtctgcagg ctcaaagagc agcgagaagc gttcagagga aagcgatccc gtgccacctt 5521 ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg atgcgggggg agcgccggac 5581 cggagcggag ccccgggcgg ctcgctgctg ccccctagcg ggggagggac gtaattacat 5641 ccctgggggc tttggggggg ggctgtccct gatatctata acaagaaaat atatatataa 5701 taagttatca cgtaagtaga acatgaaata acaatataat tatcgtatga gttaaatctt 5761 aaaagtcacg taaaagataa tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa 5821 atcagtgaca cttaccgcat tgacaagcac gcctcacggg agctccaagc ggcgactgag 5881 atgtcctaaa tgcacagcga cggattcgcg ctatttagaa agagagagca atatttcaag 5941 aatgcatgcg tcaattttac gcagactatc tttctagggt taatctagct gcatcaggat 6001 catatcgtcg ggtctttttt ccggctcagt catcgcccaa gctggcgcta tctgggcatc 6061 ggggaggaag aagcccgtgc cttttcccgc gaggttgaag cggcatggaa agagtttgcc 6121 gaggatgact gctgctgcat tgacgttgag cgaaaacgca cgtttaccat gatgattcgg 6181 gaaggtgtgg ccatgcacgc ctttaacggt gaactgttcg ttcaggccac ctgggatacc 6241 agttcgtcgc ggcttttccg gacacagttc cggatggtca gcccgaagcg catcagcaac 6301 ccgaacaata ccggcgacag ccggaactgc cgtgccggtg tgcagattaa tgacagcggt 6361 gcggcgctgg gatattacgt cagcgaggac gggtatcctg gctggatgcc gcagaaatgg 6421 acatggatac cccgtgagtt acccggcggg cgcgcttggc gtaatcatgg tcatagctgt 6481 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 6541 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 6601 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 6661 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 6721 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 6781 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 6841 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 6901 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 6961 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 7021 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 7081 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 7141 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 7201 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 7261 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 7321 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 7381 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 7441 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 7501 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 7561 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 7621 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 7681 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 7741 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 7801 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 7861 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 7921 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 7981 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 8041 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 8101 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 8161 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 8221 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 8281 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 8341 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 8401 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 8461 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 8521 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 8581 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat 8641 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga 8701 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc 8761 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac 8821 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc 8881 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg 8941 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag 9001 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc 9061 gccgctacag ggcgcgt //