LOCUS Exported 9080 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid with 4xMx PylT cassette and MX1201 PylRS for amber suppression; for transient or stable piggyBac-mediated integration. ACCESSION . VERSION . KEYWORDS pAS_4xMx PylT_FLAG-Mx PylRS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9080) AUTHORS Meineke B, Heimgartner J, Lafranchi L, Elsasser SJ TITLE Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells. JOURNAL ACS Chem Biol. 2018 Sep 27. doi: 10.1021/acschembio.8b00571. PUBMED 30260624 REFERENCE 2 (bases 1 to 9080) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9080 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(1..105) /gene="bla" /label=AmpR promoter rep_origin complement(131..586) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(268..289) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 480..499 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 713..735 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 727..744 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 728..744 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" repeat_region 874..936 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" promoter 2740..2951 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" LTR 2964..3232 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1" CDS 3280..3303 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" misc_feature 4153..4725 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4320..4337) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 4547..4566 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 4734..5528 /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 VLFVKTDLSGALNELQDEAARLSWLAATGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4788..4807) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5398..5417 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(5588..5608) /label=WPRE-R /note="WPRE, reverse primer" polyA_signal 5650..5771 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5687..5706) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5741..5760 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" repeat_region 6534..6568 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(7059..7075) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7059..7075) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(7072..7094) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 7083..7099 /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(7107..7137) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7152..7173 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(7290..7307) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7461..8049) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7541..7560) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(8220..9080) /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 8843..8862 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc 601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg 781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg 841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg 901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag 961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata 1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa 1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag 1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc 1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg 1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga 1321 cacctggggg gatacgggga aaaggcctcc acggccaact agtaaaaacc gcacttgtcc 1381 gagagaccgg ggcgtcgaac cccgctggct aggttttaga gacccgctgg tcgccggacc 1441 gtcccccgag gtacccaggc ggcgcacaag ctatataaac ctgaaggaaa tctcaacttt 1501 acacttaggt caagttactt atcgtactag agcttcagca ggaaatttaa ctaaaatcta 1561 atttaaccag catagcaaat atcatttatt cccaaaatgc taaagtttga gataaacgga 1621 cttgatttcc ggctgttttg acactatcca gaatgccttg cagatgggtg gggcatgcta 1681 aatactgcag gcaaaaaaac cgcacttgtc cgagagaccg gggcgtcgaa ccccgctggc 1741 taggttttag agacccgctg gtcgccggac cgtcccccga ggtacccagg cggcgcacaa 1801 gctatataaa cctgaaggaa atctcaactt tacacttagg tcaagttact tatcgtacta 1861 gagcttcagc aggaaattta actaaaatct aatttaacca gcatagcaaa tatcatttat 1921 tcccaaaatg ctaaagtttg agataaacgg acttgatttc cggctgtttt gacactatcc 1981 agaatgcctt gcagatgggt ggggcatgct aaatactgca gcaagaaaaa ccgcacttgt 2041 ccgagagacc ggggcgtcga accccgctgg ctaggtttta gagacccgct ggtcgccgga 2101 ccgtcccccg aggtacccag gcggcgcaca agctatataa acctgaagga aatctcaact 2161 ttacacttag gtcaagttac ttatcgtact agagcttcag caggaaattt aactaaaatc 2221 taatttaacc agcatagcaa atatcattta ttcccaaaat gctaaagttt gagataaacg 2281 gacttgattt ccggctgttt tgacactatc cagaatgcct tgcagatggg tggggcatgc 2341 taaatactgc agcgataaaa accgcacttg tccgagagac cggggcgtcg aaccccgctg 2401 gctaggtttt agagacccgc tggtcgccgg accgtccccc gaggtaccca ggcggcgcac 2461 aagctatata aacctgaagg aaatctcaac tttacactta ggtcaagtta cttatcgtac 2521 tagagcttca gcaggaaatt taactaaaat ctaatttaac cagcatagca aatatcattt 2581 attcccaaaa tgctaaagtt tgagataaac ggacttgatt tccggctgtt ttgacactat 2641 ccagaatgcc ttgcagatgg gtggggcatg ctaaatactg cagctccaag gccactagtg 2701 ctctagaaag gatctgcgat cgctccggtg cccgtcagtg ggcagagcgc acatcgccca 2761 cagtccccga gaagttgggg ggaggggtcg gcaattgaac gggtgcctag agaaggtggc 2821 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 2881 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 2941 ccagaacaca gctgaagctt cgaggggctc gcatctctcc ttcacgcgcc cgccgcccta 3001 cctgaggccg ccatccacgc cggttgagtc gcgttctgcc gcctcccgcc tgtggtgcct 3061 cctgaactgc gtccgccgtc taggtaagtt taaagctcag gtcgagaccg ggcctttgtc 3121 cggcgctccc ttggagccta cctagactca gccggctctc cacgctttgc ctgaccctgc 3181 ttgctcaact ctacgtcttt gtttcgtttt ctgttctgcg ccgttacaga tccaagctgt 3241 gaccggcgcc tactctagag ctagctcgcg accaccatgg actacaagga cgacgacgac 3301 aagacggtaa agtatacgga tgcacagata cagcgccttc gcgaatacgg caacgggacc 3361 tatgagcaga aggtcttcga ggacctcgca tcgagggatg ctgccttctc caaggagatg 3421 tccgtcgcct ctaccgacaa cgagaagaag atcaagggga tgatcgccaa tccgtcccgt 3481 catggattga cccagctgat gaacgacatc gccgacgcat tggtcgccga gggtttcatc 3541 gaggtccgta cgcccatatt catatcgaag gatgcgctgg cacgtatgac catcaccgag 3601 gacaagcccc ttttcaagca ggtcttctgg atcgacgaga aaagggcgct caggcctatg 3661 ctggcaccta acctttattc cgtcatgagg gacctgaggg accatacgga cggtccggtg 3721 aagatcttcg agatgggttc ctgcttcagg aaggagtccc acagcgggat gcatctggag 3781 gagttcacca tgctgaacct cgtggacatg ggtccccgcg gagacgccac ggaggtcctg 3841 aagaactaca tatcggtcgt gatgaaggcg gccggtctcc cggactacga cctcgtacag 3901 gaggagtccg acgtatacaa ggagaccata gacgtggaga tcaacggtca ggaggtctgt 3961 tccgcagccg tcggtccaca ctatctcgat gcggcccacg atgtccacga gccttggtcc 4021 ggagcgggat tcggtctcga acgcctgctg accatcaggg agaagtacag caccgtgaag 4081 aagggaggag ccagcatcag ctacctcaac ggtgcgaaga tcaactgatg aatcgatgga 4141 tccgcggccg cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga 4201 ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa 4261 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc 4321 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc 4381 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg 4441 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca 4501 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag 4561 cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct 4621 ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc 4681 cccgaaccac ggggacgtgg ttttcctttg aaaaacacga taatacctcc ggaatgattg 4741 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 4801 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 4861 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg 4921 aggcagcgcg gctatcgtgg ctggccgcga cgggcgttcc ttgcgcagct gtgctcgacg 4981 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 5041 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 5101 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 5161 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 5221 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg 5281 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 5341 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 5401 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 5461 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 5521 tcttctgagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc 5581 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg 5641 cgttaactaa acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 5701 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 5761 aatgtatctt atcatgtctg gaattgactc aaatgatgtc aattagtcta tcagaagctc 5821 atctggtctc ccttccgggg gacaagacat ccctgtttaa tatttaaaca gcagtgttcc 5881 caaactgggt tcttatatcc cttgctctgg tcaaccaggt tgcagggttt cctgtcctca 5941 caggaacgaa gtccctaaag aaacagtggc agccaggttt agccccggaa ttgactggat 6001 tcctttttta gggcccattg gtatggcttt ttccccgtat ccccccaggt gtctgcaggc 6061 tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg 6121 ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc 6181 cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct 6241 ttgggggggg gctgtccctg atatctataa caagaaaata tatatataat aagttatcac 6301 gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt 6361 aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac 6421 ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga tgtcctaaat 6481 gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt 6541 caattttacg cagactatct ttctagggtt aatctagctg catcaggatc atatcgtcgg 6601 gtcttttttc cggctcagtc atcgcccaag ctggcgctat ctgggcatcg gggaggaaga 6661 agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa gagtttgccg aggatgactg 6721 ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg atgattcggg aaggtgtggc 6781 catgcacgcc tttaacggtg aactgttcgt tcaggccacc tgggatacca gttcgtcgcg 6841 gcttttccgg acacagttcc ggatggtcag cccgaagcgc atcagcaacc cgaacaatac 6901 cggcgacagc cggaactgcc gtgccggtgt gcagattaat gacagcggtg cggcgctggg 6961 atattacgtc agcgaggacg ggtatcctgg ctggatgccg cagaaatgga catggatacc 7021 ccgtgagtta cccggcgggc gcgcttggcg taatcatggt catagctgtt tcctgtgtga 7081 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 7141 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 7201 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 7261 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 7321 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 7381 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 7441 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 7501 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 7561 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 7621 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 7681 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 7741 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 7801 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 7861 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 7921 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 7981 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 8041 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 8101 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 8161 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 8221 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 8281 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 8341 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 8401 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 8461 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 8521 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 8581 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 8641 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 8701 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 8761 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 8821 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 8881 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 8941 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 9001 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 9061 cggaaatgtt gaatactcat //