LOCUS Exported 7238 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION express Methanosarcina barkeri pyrrolysyl-tRNA synthetase/tRNA pair in mammalian cells to incorporate the photocrosslinkers DiZPK, DiZSeK, or DiZHSeC into amber codon site on a protein of interest. ACCESSION . VERSION . KEYWORDS pCMV-MbPylRS(DiZPK) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7238) AUTHORS Lin S, Yan H, Li L, Yang M, Peng B, Chen S, Li W, Chen PR TITLE Site-specific engineering of chemical functionalities on the surface of live hepatitis D virus. JOURNAL Angew Chem Int Ed Engl. 2013 Dec 23;52(52):13970-4. doi: 10.1002/anie.201305787. Epub 2013 Nov 12. PUBMED 24222614 REFERENCE 2 (bases 1 to 7238) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7238 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 63..442 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 443..646 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 597..617 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 643..667 /label=LNCX /note="Human CMV promoter, forward primer" promoter 877..895 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 877..894 /label=SP6 /note="SP6 promoter, forward primer" primer_bind 908..927 /label=pMT2-F /note="Synthetic intron, forward primer" regulatory 960..969 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind complement(2239..2256) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2245..2469 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2515..2943 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2602..2621) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2812..2833 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2952..2972) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2957..3286 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3137..3272 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3199..3218 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3335..4360 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" polyA_signal 4490..4611 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4527..4546) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4581..4600 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4660..4676) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4660..4676) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4673..4695) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4684..4700 /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(4708..4738) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4753..4774 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4891..4908) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5062..5650) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5142..5161) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5821..6681) /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 6444..6463 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6682..6786) /gene="bla" /label=AmpR promoter promoter complement(6884..7124) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(6934..6953) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(7104..7124) /label=hU6-F /note="Human U6 promoter, forward primer" ORIGIN 1 gttgtatcta tatcataata tgtacattta tattggctca tgtccaatat gaccgccatg 61 ttggcattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 121 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 181 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 241 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 301 tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc 361 ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt 421 attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata 481 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 541 ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca 601 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 661 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg 721 atccagcctc cgcggccggg aacggtgcat tggaacgcgg attccccgtg ccaagagtga 781 cgtaagtacc gcctatagag tctataggcc cacccccttg gcttcgttag aacgcggcta 841 caattaatac ataaccttat gtatcataca catacgattt aggtgacact atagaataac 901 atccactttg cctttctctc cacaggtgtc cactcccagg tccaactgca cggaagcttg 961 ccaccatgga taaaaaacca ttagatgttt taatatctgc gaccgggctc tggatgtcca 1021 ggactggcac gctccacaaa atcaagcacc atgaggtctc aagaagtaaa atatacattg 1081 aaatggcgtg tggagaccat cttgttgtga ataattccag gagttgtaga acagccagag 1141 cattcagaca tcataagtac agaaaaacct gcaaacgatg tagggtttcg gacgaggata 1201 tcaataattt tctcacaaga tcaaccgaaa gcaaaaacag tgtgaaagtt agggtagttt 1261 ctgctccaaa ggtcaaaaaa gctatgccga aatcagtttc aagggctccg aagcctctgg 1321 aaaattctgt ttctgcaaag gcatcaacga acacatccag atctgtacct tcgcctgcaa 1381 aatcaactcc aaattcgtct gttcccgcat cggctcctgc tccttcactt acaagaagcc 1441 agcttgatag ggttgaggct ctcttaagtc cagaggataa aatttctcta aatatggcaa 1501 agcctttcag ggaacttgag cctgaacttg tgacaagaag aaaaaacgat tttcagcggc 1561 tctataccaa tgatagagaa gactacctcg gtaaactcga acgtgatatt acgaaatttt 1621 tcgtagaccg gggttttctg gagataaagt ctcctatcct tattccggcg gaatacgtgg 1681 agagaatggg tattaataat gatactgaac tttcaaaaca gatcttccgg gtggataaaa 1741 atctctgctt gaggccaatg cttgccccga ctctgtataa ctatgcgcga aaactcgata 1801 ggattttacc aggcccaata aaaattttcg aagtcggacc ttgttaccgg aaagagtctg 1861 acggcaaaga gcacctggaa gaatttacta tggtgaactt cagtcagatg ggttcgggat 1921 gtactcggga aaatcttgaa gctctcatca aagagtttct ggactatctg gaaatcgact 1981 tcgaaatcgt aggagattcc tgtatggtct ttggggatac tcttgatata atgcacgggg 2041 acctggagct ttcttcggca gtcgtcgggc cagtttctct tgatagagaa tggggtattg 2101 acaaaccatg gataggtgca ggttttggtc ttgaacgctt gctcaaggtt atgcacggct 2161 ttaaaaacat taagagggca tcaaggtccg aatcttacta taatgggatt tcaaccaatc 2221 tataaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 2281 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 2341 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 2401 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg 2461 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc 2521 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 2581 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 2641 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 2701 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 2761 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 2821 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 2881 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 2941 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg 3001 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg 3061 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc 3121 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca 3181 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt 3241 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc 3301 ttgtatatcc attttcggat ctgatcagca cgtgatgaaa aagcctgaac tcaccgcgac 3361 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc 3421 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg 3481 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc 3541 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta 3601 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc 3661 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca 3721 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga 3781 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac 3841 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc 3901 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg 3961 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt 4021 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt 4081 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat 4141 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc 4201 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat 4261 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg 4321 aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtgctac gagatttcga 4381 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg 4441 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat 4501 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 4561 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 4621 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg 4681 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 4741 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 4801 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 4861 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 4921 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 4981 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 5041 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 5101 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 5161 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 5221 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 5281 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 5341 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 5401 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 5461 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 5521 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 5581 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 5641 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 5701 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 5761 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 5821 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 5881 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 5941 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 6001 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 6061 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 6121 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 6181 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 6241 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 6301 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 6361 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 6421 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 6481 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 6541 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 6601 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 6661 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 6721 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 6781 tccgcgcaca tttccccgaa aaatggcgga aaccccggga atctaacccg gctgaacgga 6841 tttagagtcc attcgatcta catgatcagg tttccggtgt ttcgtccttt ccacaagata 6901 tataaagcca agaaatcgaa atactttcaa gttacggtaa gcatatgata gtccatttta 6961 aaacataatt ttaaaactgc aaactaccca agaaattatt actttctacg tcacgtattt 7021 tgtactaata tctttgtgtt tacagtcaaa ttaattctaa ttatctctct aacagccttg 7081 tatcgtatat gcaaatatga aggaatcatg ggaaataggc cctcttcctg cccgaccttc 7141 gcgatgtacg ggccagatat acgccaatat tggccattag ccatattatt cattggttat 7201 atagcataaa tcaatattgg ctattggcca ttgcatac //