LOCUS       sequence-310415-        8603 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid with 4xMma PylT (M15 mutant, UUA anticodon) cassette and 
            ochre suppression reporter sfGFP 150 TAA stop, for transient 
            transfection or stable, piggybac-mediated, integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8603)
  AUTHORS   Meineke B, Heimgartner J, Eirich J, Landreh M, Elsasser SJ
  TITLE     Site-Specific Incorporation of Two ncAAs for Two-Color Bioorthogonal
            Labeling and Crosslinking of Proteins on Live Mammalian Cells.
  JOURNAL   Cell Rep. 2020 Jun 23;31(12):107811. doi: 
            10.1016/j.celrep.2020.107811.
  PUBMED    32579937
REFERENCE   2  (bases 1 to 8603)
  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: "Cell Rep.";
            date: "2020-06-23"; volume: "31(12):107811. doi"; pages: " 
            10.1016/j.celrep.2020.107811"
COMMENT     Sequence Label: pAS_4xU6-PylT M15 (UUA) sfGFP 150TAA
FEATURES             Location/Qualifiers
     source          1..8603
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     127..149
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     141..158
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     142..158
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   288..350
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(870..1118)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(1208..1456)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(1546..1794)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(1884..2132)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        2195..2406
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2419..2687
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      2721..2730
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(3462..4036,4037..4039,4040..4048)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3629..3646)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3856..3875
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4061..4459
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(4525..4545)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4587..4708
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4624..4643)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4678..4697
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5471..5505
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5996..6012)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5996..6012)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6009..6031)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6020..6036
                     /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(6044..6050,6051..6068,6069..6074))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6089..6110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6227..6244)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6398..6986)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6478..6497)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7157..7948,7949..8017))
                     /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     7780..7799
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8018..8122)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8148..8603)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8285..8306)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8497..8516
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
       61 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      121 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcctcg ttcattcacg
      181 tttttgaacc cgtggaggac gggcagactc gcggtgcaaa tgtgttttac agcgtgatgg
      241 agcagatgaa gatgctcgac acgctgcaga acacgcagct agattaaccc tagaaagata
      301 atcatattgt gacgtacgtt aaagataatc atgtgtaaaa ttgacgcatg tgttttatcg
      361 gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat tatatttaca
      421 cttacatact aataataaat tcaacaaaca atttatttat gtttatttat ttattaaaaa
      481 aaacaaaaac tcaaaatttc ttctataaag taacaaaact tttatgaggg acagcccccc
      541 cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc agcgagccgc
      601 ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga
      661 cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt ctcgctgctc
      721 tttgagcctg cagacacctg gggggatacg gggaaaaggc ctccacggcc aactagtgag
      781 acgtaaaaac cgcacttgtc cggaaacccc gggaatcgaa cccggctgaa cggatttaaa
      841 gtccgttcgg tctccctgac caggtttccg gtgtttcgtc ctttccacaa gatatataaa
      901 gccaagaaat cgaaatactt tcaagttacg gtaagcatat gatagtccat tttaaaacat
      961 aattttaaaa ctgcaaacta cccaagaaat tattactttc tacgtcacgt attttgtact
     1021 aatatctttg tgtttacagt caaattaatt ctaattatct ctctaacagc cttgtatcgt
     1081 atatgcaaat atgaaggaat catgggaaat aggccctcgc aaaaaaaccg cacttgtccg
     1141 gaaaccccgg gaatcgaacc cggctgaacg gatttaaagt ccgttcggtc tccctgacca
     1201 ggtttccggt gtttcgtcct ttccacaaga tatataaagc caagaaatcg aaatactttc
     1261 aagttacggt aagcatatga tagtccattt taaaacataa ttttaaaact gcaaactacc
     1321 caagaaatta ttactttcta cgtcacgtat tttgtactaa tatctttgtg tttacagtca
     1381 aattaattct aattatctct ctaacagcct tgtatcgtat atgcaaatat gaaggaatca
     1441 tgggaaatag gccctccaag aaaaaccgca cttgtccgga aaccccggga atcgaacccg
     1501 gctgaacgga tttaaagtcc gttcggtctc cctgaccagg tttccggtgt ttcgtccttt
     1561 ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa gcatatgata
     1621 gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt actttctacg
     1681 tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattctaa ttatctctct
     1741 aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaaataggc cctccgataa
     1801 aaaccgcact tgtccggaaa ccccgggaat cgaacccggc tgaacggatt taaagtccgt
     1861 tcggtctccc tgaccaggtt tccggtgttt cgtcctttcc acaagatata taaagccaag
     1921 aaatcgaaat actttcaagt tacggtaagc atatgatagt ccattttaaa acataatttt
     1981 aaaactgcaa actacccaag aaattattac tttctacgtc acgtattttg tactaatatc
     2041 tttgtgttta cagtcaaatt aattctaatt atctctctaa cagccttgta tcgtatatgc
     2101 aaatatgaag gaatcatggg aaataggccc tcctccaagc ctagggccac tagtgctcta
     2161 gaaaggatct gcgatcgctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc
     2221 cccgagaagt tggggggagg ggtcggcaat tgaacgggtg cctagagaag gtggcgcggg
     2281 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     2341 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     2401 acacagctga agcttcgagg ggctcgcatc tctccttcac gcgcccgccg ccctacctga
     2461 ggccgccatc cacgccggtt gagtcgcgtt ctgccgcctc ccgcctgtgg tgcctcctga
     2521 actgcgtccg ccgtctaggt aagtttaaag ctcaggtcga gaccgggcct ttgtccggcg
     2581 ctcccttgga gcctacctag actcagccgg ctctccacgc tttgcctgac cctgcttgct
     2641 caactctacg tctttgtttc gttttctgtt ctgcgccgtt acagatccaa gctgtgaccg
     2701 gcgcctactc tagagctagc gccaccatgg tgtccaaggg cgaggaactg ttcaccggcg
     2761 tggtgcccat cctggtggaa ctggatggcg acgtgaacgg ccacaagttc tctgtgcggg
     2821 gagagggcga aggcgacgcc acaaatggca agctgaccct gaagttcatc tgcaccaccg
     2881 gcaagctgcc cgtgccttgg cctaccctcg tgaccacact gacctacggc gtgcagtgct
     2941 tcagcagata ccccgaccat atgaagcggc acgacttctt caagagcgcc atgcccgagg
     3001 gctacgtgca ggaacggacc atcagcttca aggacgacgg cacctacaag accagagccg
     3061 aagtgaagtt cgagggcgac accctcgtga accggatcga gctgaagggc atcgatttca
     3121 aagaggacgg caacatcctg ggccacaagc tggagtacaa cttcaacagc cactaagtgt
     3181 acatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc cggcacaacg
     3241 tggaagatgg cagcgtgcag ctggccgacc actaccagca gaacaccccc atcggagatg
     3301 gccccgtgct gctgcccgac aaccactacc tgagcaccca gagcgtgctg agcaaggacc
     3361 ccaacgagaa gcgggaccac atggtgctgc tggaattcgt gaccgccgct ggcatcaccc
     3421 acggcatgga cgagctgtac aagggcagct gagcggccgc gcccctctcc ctcccccccc
     3481 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
     3541 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     3601 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     3661 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     3721 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     3781 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     3841 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     3901 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     3961 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     4021 aaaacacgat gataatatgg ccacaaccat ggcgtccgga atggccaagc ctttgtctca
     4081 agaagaatcc accctcattg aaagagcaac ggctacaatc aacagcatcc ccatctctga
     4141 agactacagc gtcgccagcg cagctctctc tagcgacggc cgcatcttca ctggtgtcaa
     4201 tgtatatcat tttactgggg gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc
     4261 tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt
     4321 gagcccctgc ggacggtgcc gacaggtgct tctcgatctg catcctggga tcaaagccat
     4381 agtgaaggac agtgatggac agccgacggc agttgggatt cgtgaattgc tgccctctgg
     4441 ttatgtgtgg gagggctgac catgggtcga caatcaacct ctggattaca aaatttgtga
     4501 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     4561 aatgcctttg tatcatgcgt taactaaact tgtttattgc agcttataat ggttacaaat
     4621 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4681 gtttgtccaa actcatcaat gtatcttatc atgtctggaa ttgactcaaa tgatgtcaat
     4741 tagtctatca gaagctcatc tggtctccct tccgggggac aagacatccc tgtttaatat
     4801 ttaaacagca gtgttcccaa actgggttct tatatccctt gctctggtca accaggttgc
     4861 agggtttcct gtcctcacag gaacgaagtc cctaaagaaa cagtggcagc caggtttagc
     4921 cccggaattg actggattcc ttttttaggg cccattggta tggctttttc cccgtatccc
     4981 cccaggtgtc tgcaggctca aagagcagcg agaagcgttc agaggaaagc gatcccgtgc
     5041 caccttcccc gtgcccgggc tgtccccgca cgctgccggc tcggggatgc ggggggagcg
     5101 ccggaccgga gcggagcccc gggcggctcg ctgctgcccc ctagcggggg agggacgtaa
     5161 ttacatccct gggggctttg ggggggggct gtccctgata tctataacaa gaaaatatat
     5221 atataataag ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta
     5281 aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag
     5341 ttcaaaatca gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg
     5401 actgagatgt cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat
     5461 ttcaagaatg catgcgtcaa ttttacgcag actatctttc tagggttaat ctagctgcat
     5521 caggatcata tcgtcgggtc ttttttccgg ctcagtcatc gcccaagctg gcgctatctg
     5581 ggcatcgggg aggaagaagc ccgtgccttt tcccgcgagg ttgaagcggc atggaaagag
     5641 tttgccgagg atgactgctg ctgcattgac gttgagcgaa aacgcacgtt taccatgatg
     5701 attcgggaag gtgtggccat gcacgccttt aacggtgaac tgttcgttca ggccacctgg
     5761 gataccagtt cgtcgcggct tttccggaca cagttccgga tggtcagccc gaagcgcatc
     5821 agcaacccga acaataccgg cgacagccgg aactgccgtg ccggtgtgca gattaatgac
     5881 agcggtgcgg cgctgggata ttacgtcagc gaggacgggt atcctggctg gatgccgcag
     5941 aaatggacat ggataccccg tgagttaccc ggcgggcgcg cttggcgtaa tcatggtcat
     6001 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     6061 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     6121 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     6181 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     6241 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     6301 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6361 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6421 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6481 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6541 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6601 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6661 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6721 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6781 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6841 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6901 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6961 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     7021 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     7081 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     7141 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     7201 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     7261 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     7321 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7381 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7441 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7501 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7561 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7621 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7681 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7741 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7801 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7861 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7921 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7981 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     8041 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     8101 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
     8161 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     8221 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     8281 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     8341 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     8401 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     8461 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     8521 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     8581 taatgcgccg ctacagggcg cgt
//
