LOCUS       sequence-298063-        8559 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Plasmid with 4xMmaPylT cassette and amber suppression reporter sfGFP
            150 TAG stop; for transient transfection or stable piggyBac-mediated
            integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8559)
  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 8559)
  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_4xMma PylT_GFP 150TAG 6xHis
FEATURES             Location/Qualifiers
     source          1..8559
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2715..2926
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2939..3207
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      3241..3250
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3970..3987
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    join(4004..4578,4579..4581,4582..4590)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4171..4188)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4398..4417
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4603..5001
                     /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(5067..5087)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5129..5250
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5166..5185)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5220..5239
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6013..6047
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6538..6554)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6538..6554)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6551..6573)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6562..6578
                     /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(6586..6592,6593..6610,6611..6616))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6631..6652
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6769..6786)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6940..7528)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7020..7039)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7699..8490,8491..8559))
                     /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     8322..8341
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc acggccaact agtgaaaaac cgcacttgtc
     1381 cggaaacccc gggaatctaa cccggctgaa cggatttaga gtccgttcga tctacatgat
     1441 caggtttccg aggtacccag gcggcgcaca agctatataa acctgaagga aatctcaact
     1501 ttacacttag gtcaagttac ttatcgtact agagcttcag caggaaattt aactaaaatc
     1561 taatttaacc agcatagcaa atatcattta ttcccaaaat gctaaagttt gagataaacg
     1621 gacttgattt ccggctgttt tgacactatc cagaatgcct tgcagatggg tggggcatgc
     1681 taaatactgc aggaaaaagg aaaccccggg aatctaaccc ggctgaacgg atttagagtc
     1741 cgttcgatct acatgatcag gtttccgagg tacccaggcg gcgcacaagc tatataaacc
     1801 tgaaggaaat ctcaacttta cacttaggtc aagttactta tcgtactaga gcttcagcag
     1861 gaaatttaac taaaatctaa tttaaccagc atagcaaata tcatttattc ccaaaatgct
     1921 aaagtttgag ataaacggac ttgatttccg gctgttttga cactatccag aatgccttgc
     1981 agatgggtgg ggcatgctaa atactgcagg aaaaaccgca cttgtccgga aaccccggga
     2041 atctaacccg gctgaacgga tttagagtcc gttcgatcta catgatcagg tttccgaggt
     2101 acccaggcgg cgcacaagct atataaacct gaaggaaatc tcaactttac acttaggtca
     2161 agttacttat cgtactagag cttcagcagg aaatttaact aaaatctaat ttaaccagca
     2221 tagcaaatat catttattcc caaaatgcta aagtttgaga taaacggact tgatttccgg
     2281 ctgttttgac actatccaga atgccttgca gatgggtggg gcatgctaaa tactgcagga
     2341 aaaaccgcac ttgtccggaa accccgggaa tctaacccgg ctgaacggat ttagagtccg
     2401 ttcgatctac atgatcaggt ttccgaggta cccaggcggc gcacaagcta tataaacctg
     2461 aaggaaatct caactttaca cttaggtcaa gttacttatc gtactagagc ttcagcagga
     2521 aatttaacta aaatctaatt taaccagcat agcaaatatc atttattccc aaaatgctaa
     2581 agtttgagat aaacggactt gatttccggc tgttttgaca ctatccagaa tgccttgcag
     2641 atgggtgggg catgctaaat actgcagaac tagtgctcta gaaaggatct gcgatcgctc
     2701 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     2761 ggtcggcaat tgaacgggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     2821 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     2881 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagctga agcttcgagg
     2941 ggctcgcatc tctccttcac gcgcccgccg ccctacctga ggccgccatc cacgccggtt
     3001 gagtcgcgtt ctgccgcctc ccgcctgtgg tgcctcctga actgcgtccg ccgtctaggt
     3061 aagtttaaag ctcaggtcga gaccgggcct ttgtccggcg ctcccttgga gcctacctag
     3121 actcagccgg ctctccacgc tttgcctgac cctgcttgct caactctacg tctttgtttc
     3181 gttttctgtt ctgcgccgtt acagatccaa gctgtgaccg gcgcctactc tagagctagc
     3241 gccaccatgg tgtccaaggg cgaggaactg ttcaccggcg tggtgcccat cctggtggaa
     3301 ctggatggcg acgtgaacgg ccacaagttc tctgtgcggg gagagggcga aggcgacgcc
     3361 acaaatggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccttgg
     3421 cctaccctcg tgaccacact gacctacggc gtgcagtgct tcagcagata ccccgaccat
     3481 atgaagcggc acgacttctt caagagcgcc atgcccgagg gctacgtgca ggaacggacc
     3541 atcagcttca aggacgacgg cacctacaag accagagccg aagtgaagtt cgagggcgac
     3601 accctcgtga accggatcga gctgaagggc atcgatttca aagaggacgg caacatcctg
     3661 ggccacaagc tggagtacaa cttcaacagc cactaggtgt acatcaccgc cgacaagcag
     3721 aagaacggca tcaaggccaa cttcaagatc cggcacaacg tggaagatgg cagcgtgcag
     3781 ctggccgacc actaccagca gaacaccccc atcggagatg gccccgtgct gctgcccgac
     3841 aaccactacc tgagcaccca gagcgtgctg agcaaggacc ccaacgagaa gcgggaccac
     3901 atggtgctgc tggaattcgt gaccgccgct ggcatcaccc acggcatgga cgagctgtac
     3961 aagggcagcc accatcacca tcaccattaa taataaggcc gcgcccctct ccctcccccc
     4021 cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt
     4081 tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct
     4141 tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga
     4201 atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga
     4261 ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac
     4321 gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag
     4381 ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc
     4441 agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg
     4501 tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt
     4561 gaaaaacacg atgataatat ggccacaacc atggcgtccg gaatggccaa gcctttgtct
     4621 caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct
     4681 gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc
     4741 aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct
     4801 gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc
     4861 ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc
     4921 atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct
     4981 ggttatgtgt gggagggctg accatgggtc gacaatcaac ctctggatta caaaatttgt
     5041 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     5101 ttaatgcctt tgtatcatgc gttaactaaa cttgtttatt gcagcttata atggttacaa
     5161 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     5221 tggtttgtcc aaactcatca atgtatctta tcatgtctgg aattgactca aatgatgtca
     5281 attagtctat cagaagctca tctggtctcc cttccggggg acaagacatc cctgtttaat
     5341 atttaaacag cagtgttccc aaactgggtt cttatatccc ttgctctggt caaccaggtt
     5401 gcagggtttc ctgtcctcac aggaacgaag tccctaaaga aacagtggca gccaggttta
     5461 gccccggaat tgactggatt ccttttttag ggcccattgg tatggctttt tccccgtatc
     5521 cccccaggtg tctgcaggct caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt
     5581 gccaccttcc ccgtgcccgg gctgtccccg cacgctgccg gctcggggat gcggggggag
     5641 cgccggaccg gagcggagcc ccgggcggct cgctgctgcc ccctagcggg ggagggacgt
     5701 aattacatcc ctgggggctt tggggggggg ctgtccctga tatctataac aagaaaatat
     5761 atatataata agttatcacg taagtagaac atgaaataac aatataatta tcgtatgagt
     5821 taaatcttaa aagtcacgta aaagataatc atgcgtcatt ttgactcacg cggtcgttat
     5881 agttcaaaat cagtgacact taccgcattg acaagcacgc ctcacgggag ctccaagcgg
     5941 cgactgagat gtcctaaatg cacagcgacg gattcgcgct atttagaaag agagagcaat
     6001 atttcaagaa tgcatgcgtc aattttacgc agactatctt tctagggtta atctagctgc
     6061 atcaggatca tatcgtcggg tcttttttcc ggctcagtca tcgcccaagc tggcgctatc
     6121 tgggcatcgg ggaggaagaa gcccgtgcct tttcccgcga ggttgaagcg gcatggaaag
     6181 agtttgccga ggatgactgc tgctgcattg acgttgagcg aaaacgcacg tttaccatga
     6241 tgattcggga aggtgtggcc atgcacgcct ttaacggtga actgttcgtt caggccacct
     6301 gggataccag ttcgtcgcgg cttttccgga cacagttccg gatggtcagc ccgaagcgca
     6361 tcagcaaccc gaacaatacc ggcgacagcc ggaactgccg tgccggtgtg cagattaatg
     6421 acagcggtgc ggcgctggga tattacgtca gcgaggacgg gtatcctggc tggatgccgc
     6481 agaaatggac atggataccc cgtgagttac ccggcgggcg cgcttggcgt aatcatggtc
     6541 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     6601 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     6661 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     6721 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     6781 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     6841 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     6901 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6961 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     7021 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     7081 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     7141 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     7201 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     7261 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     7321 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     7381 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     7441 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     7501 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     7561 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     7621 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     7681 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     7741 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     7801 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     7861 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     7921 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7981 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     8041 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     8101 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     8161 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     8221 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     8281 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     8341 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     8401 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     8461 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     8521 aaagggaata agggcgacac ggaaatgttg aatactcat
//
