LOCUS Exported 7226 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION plasmid with amber suppression reporter sfGFP 150 TAG stop, for transient transfection or stable, piggybac-mediated, integration. ACCESSION . VERSION . KEYWORDS pAS_sfGPF 150TAG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7226) 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 7226) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7226 /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 818..1029 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" LTR 1042..1310 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1" regulatory 1344..1353 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" misc_feature 2085..2671 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2252..2269) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2479..2498 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 2684..3082 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" primer_bind complement(3148..3168) /label=WPRE-R /note="WPRE, reverse primer" polyA_signal 3210..3331 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3247..3266) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3301..3320 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" repeat_region 4094..4128 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(4619..4635) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4619..4635) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4632..4654) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4643..4659 /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(4667..4697) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4712..4733 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4850..4867) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5021..5609) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5101..5120) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5780..6640) /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 6403..6422 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6641..6745) /gene="bla" /label=AmpR promoter rep_origin complement(6771..7226) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6908..6929) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 7120..7139 /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 aactagtgct 781 ctagaaagga tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca 841 gtccccgaga agttgggggg aggggtcggc aattgaacgg gtgcctagag aaggtggcgc 901 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga 961 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc 1021 agaacacagc tgaagcttcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc 1081 tgaggccgcc atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc 1141 tgaactgcgt ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg 1201 gcgctccctt ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt 1261 gctcaactct acgtctttgt ttcgttttct gttctgcgcc gttacagatc caagctgtga 1321 ccggcgccta ctctagagct agcgccacca tggtgtccaa gggcgaggaa ctgttcaccg 1381 gcgtggtgcc catcctggtg gaactggatg gcgacgtgaa cggccacaag ttctctgtgc 1441 ggggagaggg cgaaggcgac gccacaaatg gcaagctgac cctggagttc atctgcacca 1501 ccggcaagct gcccgtgcct tggcctaccc tcgtgaccac actgacctac ggcgtgcagt 1561 gcttcagcag ataccccgac catatgaagc ggcacgactt cttcaagagc gccatgcccg 1621 agggctacgt gcaggaacgg accatcagct tcaaggacga cggcacctac aagaccagag 1681 ccgaagtgaa gttcgagggc gacaccctcg tgaaccggat cgagctgaag ggcatcgatt 1741 tcaaagagga cggcaacatc ctgggccaca agctggagta caacttcaac agccactagg 1801 tgtacatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag atccggcaca 1861 acgtggaaga tggcagcgtg cagctggccg accactacca gcagaacacc cccatcggag 1921 atggccccgt gctgctgccc gacaaccact acctgagcac ccagagcgtg ctgagcaagg 1981 accccaacga gaagcgggac cacatggtgc tgctggaatt cgtgaccgcc gctggcatca 2041 cccacggcat ggacgagctg tacaagggca gctgagcggc cgcgcccctc tccctccccc 2101 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg 2161 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc 2221 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg 2281 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg 2341 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca 2401 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata 2461 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc 2521 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt 2581 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt 2641 tgaaaaacac gatgataata tggccacaac catggcgtcc ggaatggcca agcctttgtc 2701 tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc 2761 tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt 2821 caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc 2881 tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat 2941 cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc 3001 catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc 3061 tggttatgtg tgggagggct gaccatgggt cgacaatcaa cctctggatt acaaaatttg 3121 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc 3181 tttaatgcct ttgtatcatg cgttaactaa acttgtttat tgcagcttat aatggttaca 3241 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 3301 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gaattgactc aaatgatgtc 3361 aattagtcta tcagaagctc atctggtctc ccttccgggg gacaagacat ccctgtttaa 3421 tatttaaaca gcagtgttcc caaactgggt tcttatatcc cttgctctgg tcaaccaggt 3481 tgcagggttt cctgtcctca caggaacgaa gtccctaaag aaacagtggc agccaggttt 3541 agccccggaa ttgactggat tcctttttta gggcccattg gtatggcttt ttccccgtat 3601 ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg 3661 tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga 3721 gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg 3781 taattacatc cctgggggct ttgggggggg gctgtccctg atatctataa caagaaaata 3841 tatatataat aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag 3901 ttaaatctta aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta 3961 tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg 4021 gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa 4081 tatttcaaga atgcatgcgt caattttacg cagactatct ttctagggtt aatctagctg 4141 catcaggatc atatcgtcgg gtcttttttc cggctcagtc atcgcccaag ctggcgctat 4201 ctgggcatcg gggaggaaga agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa 4261 gagtttgccg aggatgactg ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg 4321 atgattcggg aaggtgtggc catgcacgcc tttaacggtg aactgttcgt tcaggccacc 4381 tgggatacca gttcgtcgcg gcttttccgg acacagttcc ggatggtcag cccgaagcgc 4441 atcagcaacc cgaacaatac cggcgacagc cggaactgcc gtgccggtgt gcagattaat 4501 gacagcggtg cggcgctggg atattacgtc agcgaggacg ggtatcctgg ctggatgccg 4561 cagaaatgga catggatacc ccgtgagtta cccggcgggc gcgcttggcg taatcatggt 4621 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg 4681 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt 4741 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 4801 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 4861 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 4921 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 4981 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 5041 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 5101 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 5161 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 5221 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5281 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 5341 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 5401 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 5461 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 5521 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 5581 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 5641 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 5701 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 5761 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 5821 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 5881 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 5941 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 6001 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 6061 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 6121 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 6181 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 6241 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 6301 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 6361 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 6421 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 6481 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 6541 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 6601 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 6661 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 6721 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag 6781 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca 6841 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag 6901 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg 6961 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt 7021 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag 7081 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc 7141 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc 7201 gcttaatgcg ccgctacagg gcgcgt //