LOCUS       sequence-309366-        8094 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid with Mma PylRS for amber suppression in transient or stable,
            piggybac-mediated, integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8094)
  AUTHORS   Meineke B, Heimgartner J, Lafranchi L, Elsasser SJ
  TITLE     Methanomethylophilus alvus Mx1201 provides basis for mutual 
            orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in 
            mammalian cells.
  JOURNAL   ACS Chem Biol. 2018 Sep 27. doi: 10.1021/acschembio.8b00571.
  PUBMED    30260624
REFERENCE   2  (bases 1 to 8094)
  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: "ACS Chem 
            Biol. 2018 Sep 27. doi: 10.1021/acschembio.8b00571."
COMMENT     Sequence Label: pAS_FLAG-Mma PylRS
FEATURES             Location/Qualifiers
     source          1..8094
                     /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        818..1029
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1042..1310
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      1342..1351
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1351..1374
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    join(2758..3332,3333..3335,3336..3344)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2925..2942)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3152..3171
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3357..3956
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3357..3376)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3853..3873
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4016..4036)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4078..4199
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4115..4134)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4169..4188
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4962..4996
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5487..5503)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5487..5503)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5500..5522)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5511..5527
                     /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(5535..5541,5542..5559,5560..5565))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5580..5601
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5718..5735)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5889..6477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5969..5988)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6648..7439,7440..7508))
                     /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     7271..7290
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7509..7613)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7639..8094)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7776..7797)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7988..8007
                     /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 aactagtgct
      781 ctagaaagga tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
      841 gtccccgaga agttgggggg aggggtcggc aattgaacgg gtgcctagag aaggtggcgc
      901 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
      961 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     1021 agaacacagc tgaagcttcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc
     1081 tgaggccgcc atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc
     1141 tgaactgcgt ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg
     1201 gcgctccctt ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt
     1261 gctcaactct acgtctttgt ttcgttttct gttctgcgcc gttacagatc caagctgtga
     1321 ccggcgccta ctctagagct agccaccatg gactacaagg acgacgacga caagatggac
     1381 aagaagcccc tgaacaccct gatcagcgcc acaggactgt ggatgtccag aaccggcacc
     1441 atccacaaga tcaagcacca cgaggtgtcc cggtccaaaa tctacatcga gatggcctgc
     1501 ggcgatcacc tggtcgtcaa caacagcaga agcagccgga cagccagagc cctgcggcac
     1561 cacaagtaca gaaagacctg caagcggtgc agagtgtccg acgaggacct gaacaagttc
     1621 ctgaccaagg ccaacgagga ccagaccagc gtgaaagtga aggtggtgtc cgcccccacc
     1681 cggaccaaga aagccatgcc caagagcgtg gccagagccc ccaagcccct ggaaaacacc
     1741 gaagccgctc aggcccagcc cagcggcagc aagttcagcc ccgccatccc cgtgtctacc
     1801 caggaaagcg tcagcgtccc cgccagcgtg tccaccagca tctctagcat ctcaaccggc
     1861 gccacagctt ctgccctggt caagggcaac accaacccca tcaccagcat gtctgcccct
     1921 gtgcaggcct ctgccccagc cctgaccaag tcccagaccg accggctgga agtgctcctg
     1981 aaccccaagg acgagatcag cctgaacagc ggcaagccct tccgggagct ggaaagcgag
     2041 ctgctgagcc ggcggaagaa ggacctccag caaatctacg ccgaggaacg ggagaactac
     2101 ctgggcaagc tggaaagaga gatcacccgg ttcttcgtgg accggggctt cctggaaatc
     2161 aagagcccca tcctgatccc cctggagtac atcgagcgga tgggcatcga caacgacacc
     2221 gagctgagca agcagatttt ccgggtggac aagaacttct gcctgcggcc catgctggcc
     2281 cccaacctgt acaactacct gcggaaactg gatcgcgctc tgcccgaccc catcaagatt
     2341 ttcgagatcg gcccctgcta ccggaaagag agcgacggca aagagcacct ggaagagttt
     2401 acaatgctga acttttgcca gatgggcagc ggctgcacca gagagaacct ggaatccatc
     2461 atcaccgact ttctgaacca cctggggatc gacttcaaga tcgtgggcga cagctgcatg
     2521 gtgtacggcg acaccctgga cgtgatgcac ggcgacctgg aactgtctag cgccgtcgtg
     2581 ggacccatcc ctctggaccg ggagtggggc atcgataagc cctggatcgg agccggcttc
     2641 ggcctggaac ggctgctgaa agtcaagcac gactttaaga acatcaagcg ggctgccaga
     2701 agcgagagct actacaacgg catcagcacc aacctgtgat gaggatccgc ggccgcgccc
     2761 ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc
     2821 gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa
     2881 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat
     2941 gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac
     3001 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg
     3061 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt
     3121 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg
     3181 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac
     3241 atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga
     3301 cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggcg tccggaatga
     3361 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     3421 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     3481 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     3541 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     3601 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     3661 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     3721 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     3781 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     3841 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     3901 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgagtcg
     3961 acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     4021 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgcg ttaactaaac
     4081 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4141 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4201 catgtctgga attgactcaa atgatgtcaa ttagtctatc agaagctcat ctggtctccc
     4261 ttccggggga caagacatcc ctgtttaata tttaaacagc agtgttccca aactgggttc
     4321 ttatatccct tgctctggtc aaccaggttg cagggtttcc tgtcctcaca ggaacgaagt
     4381 ccctaaagaa acagtggcag ccaggtttag ccccggaatt gactggattc cttttttagg
     4441 gcccattggt atggcttttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc
     4501 gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc
     4561 acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc
     4621 gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc
     4681 tgtccctgat atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca
     4741 tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca
     4801 tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga
     4861 caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg
     4921 attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca
     4981 gactatcttt ctagggttaa tctagctgca tcaggatcat atcgtcgggt cttttttccg
     5041 gctcagtcat cgcccaagct ggcgctatct gggcatcggg gaggaagaag cccgtgcctt
     5101 ttcccgcgag gttgaagcgg catggaaaga gtttgccgag gatgactgct gctgcattga
     5161 cgttgagcga aaacgcacgt ttaccatgat gattcgggaa ggtgtggcca tgcacgcctt
     5221 taacggtgaa ctgttcgttc aggccacctg ggataccagt tcgtcgcggc ttttccggac
     5281 acagttccgg atggtcagcc cgaagcgcat cagcaacccg aacaataccg gcgacagccg
     5341 gaactgccgt gccggtgtgc agattaatga cagcggtgcg gcgctgggat attacgtcag
     5401 cgaggacggg tatcctggct ggatgccgca gaaatggaca tggatacccc gtgagttacc
     5461 cggcgggcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5521 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5581 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5641 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5701 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5761 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5821 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5881 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5941 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     6001 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6061 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     6121 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6181 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6241 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6301 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     6361 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6421 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6481 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6541 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6601 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6661 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6721 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6781 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6841 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6901 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6961 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     7021 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     7081 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     7141 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7201 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7261 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7321 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7381 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7441 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7501 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7561 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7621 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     7681 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     7741 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     7801 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     7861 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     7921 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     7981 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     8041 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgt
//
