LOCUS       sequence-363047-        6932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes codon-optimized AF mutant of M. mazei-derived pyrrolysine 
            (Pyl) tRNA synthetase fused to a nuclear export signal (NESPylRSAF).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6932)
  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 6932)
  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)
FEATURES             Location/Qualifiers
     source          1..6932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(48..839,840..908))
                     /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     671..690
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(909..1013)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(1137..1385)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1195..1214)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1365..1385)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     enhancer        1460..1839
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1840..2043
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1994..2014
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2088..2107
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2088..2106
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2126..2135
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2156..2182
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(3567..3584)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3573..3797
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3843..4271
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3930..3949)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4140..4161
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4280..4300)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4285..4614
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4465..4600
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4527..4546
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4681..5475
                     /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(4735..4754)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5345..5364
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5649..5770
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5686..5705)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5740..5759
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5819..5835)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5819..5835)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5832..5854)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5843..5859
                     /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(5867..5873,5874..5891,5892..5897))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5912..5933
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6050..6067)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6221..6809)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6301..6320)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
       61 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
      121 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
      181 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
      241 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
      301 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
      361 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
      421 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
      481 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
      541 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
      601 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
      661 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
      721 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      781 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      841 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      901 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      961 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     1021 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc taaaaaacgg aaaccccggg
     1081 aatctaaccc ggctgaacgg atttagagtc cattcgatct acatgatcag gtttccggtg
     1141 tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca agttacggta
     1201 agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc aagaaattat
     1261 tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa attaattcca
     1321 attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat gggaaatagg
     1381 ccctccaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     1441 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
     1501 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     1561 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     1621 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     1681 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     1741 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     1801 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     1861 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     1921 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     1981 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     2041 gctctctggc taactagaga acccactgct tactggctta tcgaaattaa tacgactcac
     2101 tatagggaga cccaagctgg ctagcgccac catggcttgc cctgtgcctc tgcagctgcc
     2161 tcccctggaa cgcctgaccc tggacgataa gaaaccactg aacaccctga ttagcgccac
     2221 cggcctgtgg atgtccagga ccggcacaat ccacaagatc aagcaccacg aggtgagcag
     2281 atccaaaatc tacatcgaga tggcctgcgg cgaccacctg gtggtgaaca acagcaggag
     2341 cagcaggacc gcccgggccc tgaggcacca caagtatcgc aagacatgca agcggtgtcg
     2401 cgtgtccgac gaggatctga acaagttcct gaccaaggcc aatgaggatc agacatccgt
     2461 gaaggtgaag gtggtgtctg ccccaacccg gacaaagaag gcaatgccaa agagcgtggc
     2521 cagggccccc aagcctctgg agaacaccga ggcagcacag gcacagccat ctggcagcaa
     2581 gtttagccca gcaatccccg tgagcaccca ggagtccgtg tctgtgcctg ccagcgtgtc
     2641 cacatctatc tctagcatct ctaccggagc aacagccagc gccctggtga agggcaacac
     2701 caatcctatc acaagcatgt ccgccccagt gcaggccagc gcccctgccc tgaccaagtc
     2761 ccagacagac cgcctggagg tgctgctgaa ccctaaggat gagatcagcc tgaacagcgg
     2821 caagccattc agggagctgg agtccgagct gctgtctagg agaaagaagg acctgcagca
     2881 aatctacgcc gaggagaggg agaactatct gggcaagctg gagagggaga tcaccagatt
     2941 ctttgtggat agaggctttc tggagatcaa gtccccaatc ctgatccccc tggagtacat
     3001 cgagcggatg ggcatcgaca acgatacaga gctgtctaag cagattttca gggtggacaa
     3061 gaatttttgc ctgaggccca tgctggcccc aaacctggcc aattacctga ggaagctgga
     3121 ccgcgccctg cccgacccca tcaagatttt cgagatcggc ccatgttata gaaaggagtc
     3181 tgatggcaag gagcacctgg aggagtttac catgctgaat ttttgccaga tgggctctgg
     3241 ctgtacccgg gagaacctgg agagcatcat cacagacttc ctgaatcacc tgggcatcga
     3301 ctttaagatc gtgggcgata gctgtatggt gtttggcgac acactggatg tgatgcacgg
     3361 cgatctggag ctgtcctctg ccgtggtggg acctatccca ctggacaggg agtggggcat
     3421 cgataagccc tggatcggag caggattcgg cctggagcgc ctgctgaagg tgaagcacga
     3481 ctttaagaac atcaagcggg ctgccagaag cgaaagttat tacaatggga ttagcacaaa
     3541 cctgtgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     3601 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     3661 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     3721 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     3781 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     3841 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3901 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3961 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4021 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     4081 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4141 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     4201 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     4261 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     4321 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     4381 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4441 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4501 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     4561 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     4621 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     4681 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     4741 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     4801 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     4861 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     4921 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4981 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     5041 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     5101 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     5161 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     5221 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     5281 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     5341 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     5401 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     5461 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     5521 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     5581 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5641 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5701 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5761 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     5821 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5881 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5941 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6001 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     6061 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     6121 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6181 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6241 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6301 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     6361 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6421 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6481 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6541 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6601 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6661 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6721 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6781 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6841 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6901 tcttcaccta gatcctttta aattaaaaat ga
//
