LOCUS       sequence-440860-        7797 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).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7797)
  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 7797)
  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(+)_4x(U6 tRNA M15)_CMV NESPylRS(AF)
FEATURES             Location/Qualifiers
     source          1..7797
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(1283..1531)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(1622..1870)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(1961..2209)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2257..2276)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2448..2827
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2828..3031
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2982..3002
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3076..3095
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3076..3094
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3114..3123
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3144..3170
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(4555..4572)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4561..4785
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4831..5259
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4918..4937)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5128..5149
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5268..5288)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5273..5602
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5453..5588
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5515..5534
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5669..6463
                     /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(5723..5742)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6333..6352
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6637..6758
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6674..6693)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6728..6747
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6807..6823)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6807..6823)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6820..6842)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6831..6847
                     /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(6855..6861,6862..6879,6880..6885))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6900..6921
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7038..7055)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7209..7797)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7289..7308)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctgaaaag tgccacctga
     1201 cgtcgacaaa aaacggaaac cccgggaatc gaacccggct gaacggattt agagtccgtt
     1261 cggtctccct gaccaggttt ccggtgtttc gtcctttcca caagatatat aaagccaaga
     1321 aatcgaaata ctttcaagtt acggtaagca tatgatagtc cattttaaaa cataatttta
     1381 aaactgcaaa ctacccaaga aattattact ttctacgtca cgtattttgt actaatatct
     1441 ttgtgtttac agtcaaatta attctaatta tctctctaac agccttgtat cgtatatgca
     1501 aatatgaagg aatcatggga aataggccct cttcctgccc ctcgacaaaa aacggaaacc
     1561 ccgggaatcg aacccggctg aacggattta gagtccgttc ggtctccctg accaggtttc
     1621 cggtgtttcg tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta
     1681 cggtaagcat atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa
     1741 attattactt tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa
     1801 ttctaattat ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa
     1861 ataggccctc ttcctgcccc tcgacaaaaa acggaaaccc cgggaatcga acccggctga
     1921 acggatttag agtccgttcg gtctccctga ccaggtttcc ggtgtttcgt cctttccaca
     1981 agatatataa agccaagaaa tcgaaatact ttcaagttac ggtaagcata tgatagtcca
     2041 ttttaaaaca taattttaaa actgcaaact acccaagaaa ttattacttt ctacgtcacg
     2101 tattttgtac taatatcttt gtgtttacag tcaaattaat tctaattatc tctctaacag
     2161 ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa taggccctct tcctgcccct
     2221 cgagtctaga cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     2281 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     2341 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     2401 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
     2461 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     2521 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     2581 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     2641 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     2701 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     2761 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     2821 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     2881 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     2941 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     3001 tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac ccactgctta
     3061 ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct agcgccacca
     3121 tggcttgccc tgtgcctctg cagctgcctc ccctggaacg cctgaccctg gacgataaga
     3181 aaccactgaa caccctgatt agcgccaccg gcctgtggat gtccaggacc ggcacaatcc
     3241 acaagatcaa gcaccacgag gtgagcagat ccaaaatcta catcgagatg gcctgcggcg
     3301 accacctggt ggtgaacaac agcaggagca gcaggaccgc ccgggccctg aggcaccaca
     3361 agtatcgcaa gacatgcaag cggtgtcgcg tgtccgacga ggatctgaac aagttcctga
     3421 ccaaggccaa tgaggatcag acatccgtga aggtgaaggt ggtgtctgcc ccaacccgga
     3481 caaagaaggc aatgccaaag agcgtggcca gggcccccaa gcctctggag aacaccgagg
     3541 cagcacaggc acagccatct ggcagcaagt ttagcccagc aatccccgtg agcacccagg
     3601 agtccgtgtc tgtgcctgcc agcgtgtcca catctatctc tagcatctct accggagcaa
     3661 cagccagcgc cctggtgaag ggcaacacca atcctatcac aagcatgtcc gccccagtgc
     3721 aggccagcgc ccctgccctg accaagtccc agacagaccg cctggaggtg ctgctgaacc
     3781 ctaaggatga gatcagcctg aacagcggca agccattcag ggagctggag tccgagctgc
     3841 tgtctaggag aaagaaggac ctgcagcaaa tctacgccga ggagagggag aactatctgg
     3901 gcaagctgga gagggagatc accagattct ttgtggatag aggctttctg gagatcaagt
     3961 ccccaatcct gatccccctg gagtacatcg agcggatggg catcgacaac gatacagagc
     4021 tgtctaagca gattttcagg gtggacaaga atttttgcct gaggcccatg ctggccccaa
     4081 acctggccaa ttacctgagg aagctggacc gcgccctgcc cgaccccatc aagattttcg
     4141 agatcggccc atgttataga aaggagtctg atggcaagga gcacctggag gagtttacca
     4201 tgctgaattt ttgccagatg ggctctggct gtacccggga gaacctggag agcatcatca
     4261 cagacttcct gaatcacctg ggcatcgact ttaagatcgt gggcgatagc tgtatggtgt
     4321 ttggcgacac actggatgtg atgcacggcg atctggagct gtcctctgcc gtggtgggac
     4381 ctatcccact ggacagggag tggggcatcg ataagccctg gatcggagca ggattcggcc
     4441 tggagcgcct gctgaaggtg aagcacgact ttaagaacat caagcgggct gccagaagcg
     4501 aaagttatta caatgggatt agcacaaacc tgtgagttta aacccgctga tcagcctcga
     4561 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     4621 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     4681 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     4741 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     4801 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
     4861 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     4921 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4981 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5041 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5101 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5161 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     5221 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
     5281 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     5341 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     5401 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     5461 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     5521 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     5581 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     5641 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     5701 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     5761 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     5821 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     5881 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     5941 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     6001 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     6061 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     6121 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     6181 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     6241 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     6301 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     6361 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     6421 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     6481 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     6541 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     6601 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     6661 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     6721 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     6781 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     6841 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     6901 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     6961 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     7021 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     7081 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     7141 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     7201 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     7261 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     7321 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     7381 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     7441 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     7501 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     7561 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     7621 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     7681 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     7741 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaa
//
