LOCUS       sequence-466939-        7948 bp    DNA     circular SYN 15-DEC-2025
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    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2017 Nov 21. pii: 4641905. doi: 10.1093/nar/gkx1156."
COMMENT     Sequence Label: pNEU-hMbPylRS-4xU6M15
FEATURES             Location/Qualifiers
     source          1..7948
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      667..676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1959..1976)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1965..2189
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2235..2663
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2322..2341)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2532..2553
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2672..2692)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2677..3006
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2857..2992
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2919..2938
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3073..3867
                     /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(3127..3146)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3737..3756
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4041..4162
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4078..4097)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4132..4151
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4209..4225)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4209..4225)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4222..4244)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4233..4249
                     /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(join(4257..4263,4264..4281,4282..4287))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4302..4323
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4440..4457)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4611..5199)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4691..4710)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5370..6161,6162..6230))
                     /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     5993..6012
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6231..6335)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(6445..6693)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(6784..7032)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(7123..7371)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(7462..7710)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7758..7777)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgcca ccatggacaa gaaacccctg gacgtgctga tctccgccac cggcctgtgg
      721 atgagcagaa ccggcaccct gcacaagatc aagcaccacg aggtgtccag aagcaagatc
      781 tacatcgaga tggcctgcgg cgaccacctg gtcgtgaaca acagcagaag ctgccggacc
      841 gccagagcct tccggcacca caagtacaga aagacctgca agcggtgccg ggtgtccgac
      901 gaggatatca acaacttcct gaccagaagc accgagagca agaacagcgt gaaagtgcgg
      961 gtggtgtccg cccccaaagt gaagaaagcc atgcccaaga gcgtgtccag agcccccaag
     1021 cccctggaaa attccgtgtc cgccaaggcc agcaccaaca ccagcagatc cgtgcccagc
     1081 cctgccaaga gcacccccaa tagctctgtg cctgcctctg cccctgcccc cagcctgaca
     1141 agatcccagc tggacagagt ggaagccctg ctgagccccg aggacaagat cagcctgaac
     1201 atggccaagc ccttcagaga gctggaaccc gagctcgtga cccggcggaa gaacgacttc
     1261 cagcggctgt acaccaacga cagagaggac tacctgggca agctggaacg ggacatcacc
     1321 aagttcttcg tggaccgggg cttcctggaa atcaagagcc ccatcctgat ccccgccgag
     1381 tacgtggaac ggatgggcat caacaacgac accgagctga gcaagcagat cttccgggtg
     1441 gacaaaaacc tgtgcctgag gcccatgctg gcccccaccc tggccaacta cctgcggaaa
     1501 ctggaccgga tcctgcctgg ccccatcaag atcttcgaag tgggcccctg ctaccggaaa
     1561 gagagcgacg gcaaagagca cctggaagag tttacaatgg tcaacttctg ccagatgggc
     1621 agcggctgca ccagagagaa tctggaagct ctgatcaaag agttcctgga ctatctggaa
     1681 atcgacttcg agatcgtggg cgacagctgc atggtgttcg gcgacaccct ggacatcatg
     1741 cacggcgacc tggaactgag cagcgctgtc gtgggacccg tgtccctgga tagagagtgg
     1801 ggcatcgaca agccctggat cggagccggc ttcggcctgg aaagactgct gaaagtgatg
     1861 cacggcttca agaacatcaa gcgggcctcc cgcagcgaga gctactacaa cggcatctcc
     1921 accaacctgt gagcggccgc tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     1981 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2041 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     2101 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     2161 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     2221 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2281 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2341 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2401 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2461 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2521 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2581 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2641 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     2701 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2761 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2821 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     2881 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     2941 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3001 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     3061 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     3121 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     3181 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3241 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     3301 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     3361 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     3421 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     3481 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3541 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3601 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     3661 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     3721 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     3781 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     3841 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     3901 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     3961 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4021 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4081 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4141 ccaaactcat caatgtatct tatcatgtct gtataccggt cctctagcta gagcttggcg
     4201 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4261 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4321 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4381 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4441 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4501 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4561 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4621 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4681 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4741 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4801 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4861 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4921 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4981 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5041 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5101 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5161 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5221 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5281 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5341 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5401 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5461 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5521 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5581 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5641 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5701 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5761 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5821 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5881 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5941 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6001 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6061 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6121 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6181 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6241 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6301 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6361 gacgtcgaca aaaaacggaa accccgggaa tcgaacccgg ctgaacggat ttagagtccg
     6421 ttcggtctcc ctgaccaggt ttccggtgtt tcgtcctttc cacaagatat ataaagccaa
     6481 gaaatcgaaa tactttcaag ttacggtaag catatgatag tccattttaa aacataattt
     6541 taaaactgca aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat
     6601 ctttgtgttt acagtcaaat taattctaat tatctctcta acagccttgt atcgtatatg
     6661 caaatatgaa ggaatcatgg gaaataggcc ctcttcctgc ccctcgacaa aaaacggaaa
     6721 ccccgggaat cgaacccggc tgaacggatt tagagtccgt tcggtctccc tgaccaggtt
     6781 tccggtgttt cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt
     6841 tacggtaagc atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag
     6901 aaattattac tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt
     6961 aattctaatt atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg
     7021 aaataggccc tcttcctgcc cctcgacaaa aaacggaaac cccgggaatc gaacccggct
     7081 gaacggattt agagtccgtt cggtctccct gaccaggttt ccggtgtttc gtcctttcca
     7141 caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc
     7201 cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca
     7261 cgtattttgt actaatatct ttgtgtttac agtcaaatta attctaatta tctctctaac
     7321 agccttgtat cgtatatgca aatatgaagg aatcatggga aataggccct cttcctgccc
     7381 ctcgacaaaa aacggaaacc ccgggaatcg aacccggctg aacggattta gagtccgttc
     7441 ggtctccctg accaggtttc cggtgtttcg tcctttccac aagatatata aagccaagaa
     7501 atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa
     7561 aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt
     7621 tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa
     7681 atatgaagga atcatgggaa ataggccctc ttcctgcccc tcgagtctag acccgatccc
     7741 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     7801 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     7861 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     7921 cgcgatgtac gggccagata tacgcgtt
//
