LOCUS       Exported                8136 bp ds-DNA     circular SYN 27-MAY-2022
DEFINITION  Encodes codon-optimized AF mutant of M. mazei pyrrolysine (Pyl) tRNA
            synthetase fused to a nuclear export signal (NESPylRSAF).
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3.1(+)_4x(U6 tRNA M15)_CMV NESPylRS(AF)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8136)
  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 8136)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 27, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8136
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        complement(208..448)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     promoter        complement(547..787)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     promoter        complement(886..1126)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     promoter        complement(1225..1465)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     complement(1513..1532)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1704..2083
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2084..2287
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2238..2258
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2332..2351
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2332..2350
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2370..2379
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2400..2426
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(3811..3828)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3817..4041
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4087..4515
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4174..4193)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4384..4405
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4524..4544)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4529..4858
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4709..4844
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4771..4790
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4925..5719
                     /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(4979..4998)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5589..5608
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5893..6014
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5930..5949)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5984..6003
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6063..6079)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6063..6079)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6076..6098)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6087..6103
                     /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(6111..6141)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6156..6177
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6294..6311)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6465..7053)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6545..6564)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7224..8084)
                     /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     7847..7866
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8085..53)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
       61 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tgaaaagtgc cacctgacgt
      121 cgacaaaaaa cggaaacccc gggaatcgaa cccggctgaa cggatttaga gtccgttcgg
      181 tctccctgac caggtttccg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat
      241 cgaaatactt tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa
      301 ctgcaaacta cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg
      361 tgtttacagt caaattaatt ctaattatct ctctaacagc cttgtatcgt atatgcaaat
      421 atgaaggaat catgggaaat aggccctctt cctgcccctc gacaaaaaac ggaaaccccg
      481 ggaatcgaac ccggctgaac ggatttagag tccgttcggt ctccctgacc aggtttccgg
      541 tgtttcgtcc tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg
      601 taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt
      661 attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc
      721 taattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata
      781 ggccctcttc ctgcccctcg acaaaaaacg gaaaccccgg gaatcgaacc cggctgaacg
      841 gatttagagt ccgttcggtc tccctgacca ggtttccggt gtttcgtcct ttccacaaga
      901 tatataaagc caagaaatcg aaatactttc aagttacggt aagcatatga tagtccattt
      961 taaaacataa ttttaaaact gcaaactacc caagaaatta ttactttcta cgtcacgtat
     1021 tttgtactaa tatctttgtg tttacagtca aattaattct aattatctct ctaacagcct
     1081 tgtatcgtat atgcaaatat gaaggaatca tgggaaatag gccctcttcc tgcccctcga
     1141 caaaaaacgg aaaccccggg aatcgaaccc ggctgaacgg atttagagtc cgttcggtct
     1201 ccctgaccag gtttccggtg tttcgtcctt tccacaagat atataaagcc aagaaatcga
     1261 aatactttca agttacggta agcatatgat agtccatttt aaaacataat tttaaaactg
     1321 caaactaccc aagaaattat tactttctac gtcacgtatt ttgtactaat atctttgtgt
     1381 ttacagtcaa attaattcta attatctctc taacagcctt gtatcgtata tgcaaatatg
     1441 aaggaatcat gggaaatagg ccctcttcct gcccctcgag tctagacccg atcccctatg
     1501 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     1561 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     1621 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     1681 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
     1741 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     1801 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     1861 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     1921 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     1981 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     2041 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     2101 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     2161 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     2221 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     2281 cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac
     2341 tcactatagg gagacccaag ctggctagcg ccaccatggc ttgccctgtg cctctgcagc
     2401 tgcctcccct ggaacgcctg accctggacg ataagaaacc actgaacacc ctgattagcg
     2461 ccaccggcct gtggatgtcc aggaccggca caatccacaa gatcaagcac cacgaggtga
     2521 gcagatccaa aatctacatc gagatggcct gcggcgacca cctggtggtg aacaacagca
     2581 ggagcagcag gaccgcccgg gccctgaggc accacaagta tcgcaagaca tgcaagcggt
     2641 gtcgcgtgtc cgacgaggat ctgaacaagt tcctgaccaa ggccaatgag gatcagacat
     2701 ccgtgaaggt gaaggtggtg tctgccccaa cccggacaaa gaaggcaatg ccaaagagcg
     2761 tggccagggc ccccaagcct ctggagaaca ccgaggcagc acaggcacag ccatctggca
     2821 gcaagtttag cccagcaatc cccgtgagca cccaggagtc cgtgtctgtg cctgccagcg
     2881 tgtccacatc tatctctagc atctctaccg gagcaacagc cagcgccctg gtgaagggca
     2941 acaccaatcc tatcacaagc atgtccgccc cagtgcaggc cagcgcccct gccctgacca
     3001 agtcccagac agaccgcctg gaggtgctgc tgaaccctaa ggatgagatc agcctgaaca
     3061 gcggcaagcc attcagggag ctggagtccg agctgctgtc taggagaaag aaggacctgc
     3121 agcaaatcta cgccgaggag agggagaact atctgggcaa gctggagagg gagatcacca
     3181 gattctttgt ggatagaggc tttctggaga tcaagtcccc aatcctgatc cccctggagt
     3241 acatcgagcg gatgggcatc gacaacgata cagagctgtc taagcagatt ttcagggtgg
     3301 acaagaattt ttgcctgagg cccatgctgg ccccaaacct ggccaattac ctgaggaagc
     3361 tggaccgcgc cctgcccgac cccatcaaga ttttcgagat cggcccatgt tatagaaagg
     3421 agtctgatgg caaggagcac ctggaggagt ttaccatgct gaatttttgc cagatgggct
     3481 ctggctgtac ccgggagaac ctggagagca tcatcacaga cttcctgaat cacctgggca
     3541 tcgactttaa gatcgtgggc gatagctgta tggtgtttgg cgacacactg gatgtgatgc
     3601 acggcgatct ggagctgtcc tctgccgtgg tgggacctat cccactggac agggagtggg
     3661 gcatcgataa gccctggatc ggagcaggat tcggcctgga gcgcctgctg aaggtgaagc
     3721 acgactttaa gaacatcaag cgggctgcca gaagcgaaag ttattacaat gggattagca
     3781 caaacctgtg agtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     3841 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     3901 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     3961 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     4021 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     4081 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4141 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4201 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4261 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4321 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4381 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4441 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4501 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     4561 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     4621 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4681 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     4741 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     4801 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     4861 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     4921 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     4981 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     5041 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     5101 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     5161 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     5221 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     5281 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     5341 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     5401 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     5461 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     5521 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     5581 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     5641 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     5701 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     5761 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     5821 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     5881 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5941 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     6001 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     6061 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     6121 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     6181 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6241 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     6301 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6361 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     6421 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     6481 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     6541 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     6601 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     6661 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6721 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6781 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6841 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6901 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6961 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7021 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7081 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     7141 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     7201 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     7261 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     7321 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     7381 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     7441 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     7501 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     7561 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     7621 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     7681 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     7741 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     7801 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     7861 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     7921 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7981 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     8041 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     8101 tattattgaa gcatttatca gggttattgt ctcatg
//
