LOCUS pNEU-hMbPylRS-4xU6M15 7948 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Plasmid encoding for the Pyrrolysine translational pair that enables
efficent incoporation of Pyl-like click amino acids in response to
the amber codon into proteins in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS pNEU-hMbPylRS-4xU6M15
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7948)
AUTHORS Serfling R, Lorenz C, Etzel M, Schicht G, Bottke T, Morl M, Coin I
TITLE Designer tRNAs for efficient incorporation of non-canonical amino
acids by the pyrrolysine system in mammalian cells.
JOURNAL Nucleic Acids Res. 2017 Nov 21. pii: 4641905. doi:
10.1093/nar/gkx1156.
PUBMED 29177436
REFERENCE 2 (bases 1 to 7948)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7948
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter complement(121..361)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
promoter complement(460..700)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
promoter complement(799..1039)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
promoter complement(1138..1378)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(1426..1445)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 1617..1996
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 1997..2200
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 2151..2171
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 2245..2264
/label=T7
/note="T7 promoter, forward primer"
promoter 2245..2263
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 2283..2292
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
primer_bind complement(3575..3592)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 3581..3805
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 3851..4279
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3938..3957)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4148..4169
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4288..4308)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 4293..4622
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 4473..4608
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 4535..4554
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 4689..5483
/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(4743..4762)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 5353..5372
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 5657..5778
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(5694..5713)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 5748..5767
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(5825..5841)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5825..5841)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5838..5860)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5849..5865
/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(5873..5903)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5918..5939
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(6056..6073)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6227..6815)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6307..6326)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6986..7846)
/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 7609..7628
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7847..3)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gcgcacattt ccccgaaaag tgccacctga cgtcgacaaa aaacggaaac cccgggaatc
61 gaacccggct gaacggattt agagtccgtt cggtctccct gaccaggttt ccggtgtttc
121 gtcctttcca caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca
181 tatgatagtc cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact
241 ttctacgtca cgtattttgt actaatatct ttgtgtttac agtcaaatta attctaatta
301 tctctctaac agccttgtat cgtatatgca aatatgaagg aatcatggga aataggccct
361 cttcctgccc ctcgacaaaa aacggaaacc ccgggaatcg aacccggctg aacggattta
421 gagtccgttc ggtctccctg accaggtttc cggtgtttcg tcctttccac aagatatata
481 aagccaagaa atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac
541 ataattttaa aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta
601 ctaatatctt tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc
661 gtatatgcaa atatgaagga atcatgggaa ataggccctc ttcctgcccc tcgacaaaaa
721 acggaaaccc cgggaatcga acccggctga acggatttag agtccgttcg gtctccctga
781 ccaggtttcc ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact
841 ttcaagttac ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact
901 acccaagaaa ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag
961 tcaaattaat tctaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa
1021 tcatgggaaa taggccctct tcctgcccct cgacaaaaaa cggaaacccc gggaatcgaa
1081 cccggctgaa cggatttaga gtccgttcgg tctccctgac caggtttccg gtgtttcgtc
1141 ctttccacaa gatatataaa gccaagaaat cgaaatactt tcaagttacg gtaagcatat
1201 gatagtccat tttaaaacat aattttaaaa ctgcaaacta cccaagaaat tattactttc
1261 tacgtcacgt attttgtact aatatctttg tgtttacagt caaattaatt ctaattatct
1321 ctctaacagc cttgtatcgt atatgcaaat atgaaggaat catgggaaat aggccctctt
1381 cctgcccctc gagtctagac ccgatcccct atggtgcact ctcagtacaa tctgctctga
1441 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
1501 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
1561 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
1621 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
1681 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
1741 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
1801 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
1861 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
1921 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
1981 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
2041 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
2101 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
2161 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
2221 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta
2281 gcgccaccat ggacaagaaa cccctggacg tgctgatctc cgccaccggc ctgtggatga
2341 gcagaaccgg caccctgcac aagatcaagc accacgaggt gtccagaagc aagatctaca
2401 tcgagatggc ctgcggcgac cacctggtcg tgaacaacag cagaagctgc cggaccgcca
2461 gagccttccg gcaccacaag tacagaaaga cctgcaagcg gtgccgggtg tccgacgagg
2521 atatcaacaa cttcctgacc agaagcaccg agagcaagaa cagcgtgaaa gtgcgggtgg
2581 tgtccgcccc caaagtgaag aaagccatgc ccaagagcgt gtccagagcc cccaagcccc
2641 tggaaaattc cgtgtccgcc aaggccagca ccaacaccag cagatccgtg cccagccctg
2701 ccaagagcac ccccaatagc tctgtgcctg cctctgcccc tgcccccagc ctgacaagat
2761 cccagctgga cagagtggaa gccctgctga gccccgagga caagatcagc ctgaacatgg
2821 ccaagccctt cagagagctg gaacccgagc tcgtgacccg gcggaagaac gacttccagc
2881 ggctgtacac caacgacaga gaggactacc tgggcaagct ggaacgggac atcaccaagt
2941 tcttcgtgga ccggggcttc ctggaaatca agagccccat cctgatcccc gccgagtacg
3001 tggaacggat gggcatcaac aacgacaccg agctgagcaa gcagatcttc cgggtggaca
3061 aaaacctgtg cctgaggccc atgctggccc ccaccctggc caactacctg cggaaactgg
3121 accggatcct gcctggcccc atcaagatct tcgaagtggg cccctgctac cggaaagaga
3181 gcgacggcaa agagcacctg gaagagttta caatggtcaa cttctgccag atgggcagcg
3241 gctgcaccag agagaatctg gaagctctga tcaaagagtt cctggactat ctggaaatcg
3301 acttcgagat cgtgggcgac agctgcatgg tgttcggcga caccctggac atcatgcacg
3361 gcgacctgga actgagcagc gctgtcgtgg gacccgtgtc cctggataga gagtggggca
3421 tcgacaagcc ctggatcgga gccggcttcg gcctggaaag actgctgaaa gtgatgcacg
3481 gcttcaagaa catcaagcgg gcctcccgca gcgagagcta ctacaacggc atctccacca
3541 acctgtgagc ggccgcttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
3601 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
3661 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
3721 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
3781 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
3841 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
3901 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
3961 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
4021 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
4081 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
4141 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
4201 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
4261 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
4321 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
4381 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
4441 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
4501 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
4561 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
4621 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
4681 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
4741 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
4801 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
4861 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
4921 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
4981 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
5041 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
5101 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
5161 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
5221 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
5281 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
5341 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
5401 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
5461 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
5521 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
5581 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
5641 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
5701 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
5761 actcatcaat gtatcttatc atgtctgtat accggtcctc tagctagagc ttggcgtaat
5821 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
5881 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
5941 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
6001 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
6061 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
6121 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
6181 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
6241 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
6301 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
6361 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
6421 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
6481 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
6541 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
6601 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
6661 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
6721 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
6781 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
6841 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
6901 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
6961 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
7021 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
7081 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
7141 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
7201 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
7261 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
7321 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
7381 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
7441 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
7501 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
7561 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
7621 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
7681 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
7741 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
7801 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
7861 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
7921 tttagaaaaa taaacaaata ggggttcc
//
