LOCUS       sequence-363026-        8518 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes codon-optimized AF mutant of M. mazei pyrrolysine (Pyl) tRNA
            synthetase fused to a nuclear export signal (NESPylRSAF),tRNACUAPyl.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8518)
  AUTHORS   Arsic A, Hagemann C, Stajkovic N, Schubert T, Nikic-Spiegel I
  TITLE     Minimal genetically encoded tags for fluorescent protein labeling in
            living neurons.
  JOURNAL   Nat Commun. 2022 Jan 14;13(1):314. doi: 10.1038/s41467-022-27956-y.
  PUBMED    35031604
REFERENCE   2  (bases 1 to 8518)
  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: "Nat 
            Commun."; date: "2022-01-14"; volume: "13(1):314. doi"; pages: " 
            10.1038/s41467-022-27956-y"
COMMENT     Sequence Label: pcDNA3.1(+)_U6 tRNAPyl_CMV
            NESPylRS(AF)_IRES_e...
FEATURES             Location/Qualifiers
     source          1..8518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(75..95)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        80..409
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      260..395
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     322..341
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        457..504
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     polyA_signal    1012..1133
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1049..1068)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1103..1122
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1182..1198)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1182..1198)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1195..1217)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1206..1222
                     /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(1230..1236,1237..1254,1255..1260))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1275..1296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1413..1430)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1584..2172)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1664..1683)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2343..3134,3135..3203))
                     /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     2966..2985
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3204..3308)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(3432..3680)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3490..3509)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3660..3680)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     enhancer        3755..4134
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4135..4338
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4289..4309
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4383..4402
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4383..4401
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4421..4430
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4451..4477
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     misc_feature    5867..6419
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(6011..6028)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     6238..6257
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      6444..6453
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6453..6494
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             7803..7829
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(7880..7897)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7886..8110
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(8156..8518,1..66)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8243..8262)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8453..8474
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
       61 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
      121 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
      181 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
      241 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
      301 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
      361 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
      421 agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag
      481 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt
      541 gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg
      601 ctcgggttct cccgggactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc
      661 gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg
      721 gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg
      781 ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg
      841 gccggcaact gcgtgcactt cgtggccgag gagcaggact gacacgtgct acgagatttc
      901 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
      961 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     1021 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     1081 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     1141 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     1201 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     1261 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     1321 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     1381 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     1441 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     1501 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     1561 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1621 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     1681 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1741 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     1801 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     1861 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     1921 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     1981 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     2041 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     2101 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     2161 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2221 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2281 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     2341 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     2401 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     2461 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     2521 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     2581 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     2641 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     2701 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     2761 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     2821 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     2881 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     2941 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     3001 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     3061 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     3121 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     3181 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     3241 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     3301 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctaaaa
     3361 aacggaaacc ccgggaatct aacccggctg aacggattta gagtccattc gatctacatg
     3421 atcaggtttc cggtgtttcg tcctttccac aagatatata aagccaagaa atcgaaatac
     3481 tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa aactgcaaac
     3541 tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt tgtgtttaca
     3601 gtcaaattaa ttccaattat ctctctaaca gccttgtatc gtatatgcaa atatgaagga
     3661 atcatgggaa ataggccctc caattgcatg aagaatctgc ttagggttag gcgttttgcg
     3721 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
     3781 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     3841 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     3901 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     3961 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     4021 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     4081 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     4141 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     4201 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     4261 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     4321 gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa
     4381 attaatacga ctcactatag ggagacccaa gctggctagc gccaccatgg cttgccctgt
     4441 gcctctgcag ctgcctcccc tggaacgcct gaccctggac gataagaaac cactgaacac
     4501 cctgattagc gccaccggcc tgtggatgtc caggaccggc acaatccaca agatcaagca
     4561 ccacgaggtg agcagatcca aaatctacat cgagatggcc tgcggcgacc acctggtggt
     4621 gaacaacagc aggagcagca ggaccgcccg ggccctgagg caccacaagt atcgcaagac
     4681 atgcaagcgg tgtcgcgtgt ccgacgagga tctgaacaag ttcctgacca aggccaatga
     4741 ggatcagaca tccgtgaagg tgaaggtggt gtctgcccca acccggacaa agaaggcaat
     4801 gccaaagagc gtggccaggg cccccaagcc tctggagaac accgaggcag cacaggcaca
     4861 gccatctggc agcaagttta gcccagcaat ccccgtgagc acccaggagt ccgtgtctgt
     4921 gcctgccagc gtgtccacat ctatctctag catctctacc ggagcaacag ccagcgccct
     4981 ggtgaagggc aacaccaatc ctatcacaag catgtccgcc ccagtgcagg ccagcgcccc
     5041 tgccctgacc aagtcccaga cagaccgcct ggaggtgctg ctgaacccta aggatgagat
     5101 cagcctgaac agcggcaagc cattcaggga gctggagtcc gagctgctgt ctaggagaaa
     5161 gaaggacctg cagcaaatct acgccgagga gagggagaac tatctgggca agctggagag
     5221 ggagatcacc agattctttg tggatagagg ctttctggag atcaagtccc caatcctgat
     5281 ccccctggag tacatcgagc ggatgggcat cgacaacgat acagagctgt ctaagcagat
     5341 tttcagggtg gacaagaatt tttgcctgag gcccatgctg gccccaaacc tggccaatta
     5401 cctgaggaag ctggaccgcg ccctgcccga ccccatcaag attttcgaga tcggcccatg
     5461 ttatagaaag gagtctgatg gcaaggagca cctggaggag tttaccatgc tgaatttttg
     5521 ccagatgggc tctggctgta cccgggagaa cctggagagc atcatcacag acttcctgaa
     5581 tcacctgggc atcgacttta agatcgtggg cgatagctgt atggtgtttg gcgacacact
     5641 ggatgtgatg cacggcgatc tggagctgtc ctctgccgtg gtgggaccta tcccactgga
     5701 cagggagtgg ggcatcgata agccctggat cggagcagga ttcggcctgg agcgcctgct
     5761 gaaggtgaag cacgacttta agaacatcaa gcgggctgcc agaagcgaaa gttattacaa
     5821 tgggattagc acaaacctgt gaaagcttgg taccgagctc ggatccacgt tactggccga
     5881 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg
     5941 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg
     6001 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt
     6061 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac
     6121 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca
     6181 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg
     6241 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg
     6301 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa
     6361 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatag
     6421 aattcagacc caagctggct agcgccacca tgggcaagcc tattcccaac cccctgctgg
     6481 gcctggatag caccgctgac gacccctccg ctgccgatag aaacgtggaa atctggaaga
     6541 tcaagaaact gattaagagc ctggaggctg ccagaggaaa cggcaccagc atgatctccc
     6601 tgattatccc tcccaaggat cagatcagca gggtcgctaa gatgctcgcc gatgatttcg
     6661 gcaccgccag caatatcaag agcagggtga acagactgag cgtgctgggc gccattacca
     6721 gcgtgcagca gagactgaag ctgtacaaca aggtgccccc caacggactg gtggtgtact
     6781 gtggaaccat cgtgaccgaa gagggcaagg aaaaaaaagt gaacatcgac ttcgagccct
     6841 tcaaacccat caacacctcc ctctacctgt gcgacaataa gttccacacc gaggccctga
     6901 cagctctgct cagcgacgac tccaagttcg gcttcattgt gatcgacgga agcggcgctc
     6961 tgttcggaac cctgcagggc aacaccaggg aggtgctcca caagttcacc gtggacctgc
     7021 ctaaaaagca cggcagaggc ggccagagcg ctctgagatt cgccaggctg agaatggaga
     7081 agaggcacaa ctacgtgaga aaggtggccg aaaccgccgt gcagctgttc attagcggcg
     7141 acaaggtgaa cgtggccggc ctggtgctgg ccggctccgc cgactttaag accgagctgt
     7201 cccagagcga tatgttcgat cagaggctgc aaagcaaggt gctgaagctg gtggacatca
     7261 gctacggagg cgagaacggc ttcaaccagg ccatcgaact cagcaccgag gtgctgagca
     7321 acgtgaaatt cattcaagag aagaagctca tcggcaggta cttcgacgag atcagccagg
     7381 ataccggcaa gtactgcttt ggagtggagg ataccctgaa ggccctggag atgggcgccg
     7441 tggagatcct gattgtgtac gagaatctgg acatcatgag atatgtgctc cactgccagg
     7501 gcaccgagga ggagaaaatc ctgtacctca cccccgagca ggagaaggac aaaagccact
     7561 tcaccgataa ggagaccggc caggaacacg agctgattga gtccatgccc ctgctggagt
     7621 ggtttgccaa caactacaag aaattcggcg ccaccctcga gatcgtcacc gacaagtccc
     7681 aggaaggctc ccagttcgtc aagggcttcg gcggcatcgg aggcatcctc aggtacaggg
     7741 tcgacttcca gggcatggag tatcagggcg gcgacgacga gttcttcgat ctggacgact
     7801 actacccata cgatgttcca gattacgctt aatgagcggc cgctcgagtc tagagggccc
     7861 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     7921 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     7981 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     8041 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     8101 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     8161 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     8221 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     8281 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     8341 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     8401 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     8461 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataag
//
