LOCUS       sequence-309629-        8549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid with 4xMma PylT cassette and amber suppression reporter 
            sfGFP 150 TAG 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 8549)
  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 8549)
  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 sfGPF 150TAG
FEATURES             Location/Qualifiers
     source          1..8549
                     /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        2141..2352
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2365..2633
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      2667..2676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(3408..3982,3983..3985,3986..3994)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3575..3592)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3802..3821
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4007..4405
                     /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(4471..4491)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4533..4654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4570..4589)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4624..4643
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5417..5451
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5942..5958)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5942..5958)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5955..5977)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5966..5982
                     /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(5990..5996,5997..6014,6015..6020))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6035..6056
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6173..6190)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6344..6932)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6424..6443)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7103..7894,7895..7963))
                     /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     7726..7745
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7964..8068)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8094..8549)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8231..8252)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8443..8462
                     /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 aactagtgaa
      781 aaaccgcact tgtccggaaa ccccgggaat ctaacccggc tgaacggatt tagagtccgt
      841 tcgatctaca tgatcaggtt tccgaggtac ccaggcggcg cacaagctat ataaacctga
      901 aggaaatctc aactttacac ttaggtcaag ttacttatcg tactagagct tcagcaggaa
      961 atttaactaa aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa
     1021 gtttgagata aacggacttg atttccggct gttttgacac tatccagaat gccttgcaga
     1081 tgggtggggc atgctaaata ctgcaggaaa aaccgcactt gtccggaaac cccgggaatc
     1141 taacccggct gaacggattt agagtccgtt cgatctacat gatcaggttt ccgaggtacc
     1201 caggcggcgc acaagctata taaacctgaa ggaaatctca actttacact taggtcaagt
     1261 tacttatcgt actagagctt cagcaggaaa tttaactaaa atctaattta accagcatag
     1321 caaatatcat ttattcccaa aatgctaaag tttgagataa acggacttga tttccggctg
     1381 ttttgacact atccagaatg ccttgcagat gggtggggca tgctaaatac tgcaggaaaa
     1441 accgcacttg tccggaaacc ccgggaatct aacccggctg aacggattta gagtccgttc
     1501 gatctacatg atcaggtttc cgaggtaccc aggcggcgca caagctatat aaacctgaag
     1561 gaaatctcaa ctttacactt aggtcaagtt acttatcgta ctagagcttc agcaggaaat
     1621 ttaactaaaa tctaatttaa ccagcatagc aaatatcatt tattcccaaa atgctaaagt
     1681 ttgagataaa cggacttgat ttccggctgt tttgacacta tccagaatgc cttgcagatg
     1741 ggtggggcat gctaaatact gcaggaaaaa ccgcacttgt ccggaaaccc cgggaatcta
     1801 acccggctga acggatttag agtccgttcg atctacatga tcaggtttcc gaggtaccca
     1861 ggcggcgcac aagctatata aacctgaagg aaatctcaac tttacactta ggtcaagtta
     1921 cttatcgtac tagagcttca gcaggaaatt taactaaaat ctaatttaac cagcatagca
     1981 aatatcattt attcccaaaa tgctaaagtt tgagataaac ggacttgatt tccggctgtt
     2041 ttgacactat ccagaatgcc ttgcagatgg gtggggcatg ctaaatactg cagaactagt
     2101 gctctagaaa ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc
     2161 acagtccccg agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg
     2221 cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg
     2281 ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc
     2341 gccagaacac agctgaagct tcgaggggct cgcatctctc cttcacgcgc ccgccgccct
     2401 acctgaggcc gccatccacg ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc
     2461 tcctgaactg cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt
     2521 ccggcgctcc cttggagcct acctagactc agccggctct ccacgctttg cctgaccctg
     2581 cttgctcaac tctacgtctt tgtttcgttt tctgttctgc gccgttacag atccaagctg
     2641 tgaccggcgc ctactctaga gctagcgcca ccatggtgtc caagggcgag gaactgttca
     2701 ccggcgtggt gcccatcctg gtggaactgg atggcgacgt gaacggccac aagttctctg
     2761 tgcggggaga gggcgaaggc gacgccacaa atggcaagct gaccctggag ttcatctgca
     2821 ccaccggcaa gctgcccgtg ccttggccta ccctcgtgac cacactgacc tacggcgtgc
     2881 agtgcttcag cagatacccc gaccatatga agcggcacga cttcttcaag agcgccatgc
     2941 ccgagggcta cgtgcaggaa cggaccatca gcttcaagga cgacggcacc tacaagacca
     3001 gagccgaagt gaagttcgag ggcgacaccc tcgtgaaccg gatcgagctg aagggcatcg
     3061 atttcaaaga ggacggcaac atcctgggcc acaagctgga gtacaacttc aacagccact
     3121 aggtgtacat caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccggc
     3181 acaacgtgga agatggcagc gtgcagctgg ccgaccacta ccagcagaac acccccatcg
     3241 gagatggccc cgtgctgctg cccgacaacc actacctgag cacccagagc gtgctgagca
     3301 aggaccccaa cgagaagcgg gaccacatgg tgctgctgga attcgtgacc gccgctggca
     3361 tcacccacgg catggacgag ctgtacaagg gcagctgagc ggccgcgccc ctctccctcc
     3421 ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat
     3481 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct
     3541 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg
     3601 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta
     3661 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag
     3721 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg
     3781 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat
     3841 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca
     3901 tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc
     3961 ctttgaaaaa cacgatgata atatggccac aaccatggcg tccggaatgg ccaagccttt
     4021 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat
     4081 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg
     4141 tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc
     4201 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg
     4261 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa
     4321 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc
     4381 ctctggttat gtgtgggagg gctgaccatg ggtcgacaat caacctctgg attacaaaat
     4441 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     4501 tgctttaatg cctttgtatc atgcgttaac taaacttgtt tattgcagct tataatggtt
     4561 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4621 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggaattga ctcaaatgat
     4681 gtcaattagt ctatcagaag ctcatctggt ctcccttccg ggggacaaga catccctgtt
     4741 taatatttaa acagcagtgt tcccaaactg ggttcttata tcccttgctc tggtcaacca
     4801 ggttgcaggg tttcctgtcc tcacaggaac gaagtcccta aagaaacagt ggcagccagg
     4861 tttagccccg gaattgactg gattcctttt ttagggccca ttggtatggc tttttccccg
     4921 tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc
     4981 ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg
     5041 ggagcgccgg accggagcgg agccccgggc ggctcgctgc tgccccctag cgggggaggg
     5101 acgtaattac atccctgggg gctttggggg ggggctgtcc ctgatatcta taacaagaaa
     5161 atatatatat aataagttat cacgtaagta gaacatgaaa taacaatata attatcgtat
     5221 gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt cattttgact cacgcggtcg
     5281 ttatagttca aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa
     5341 gcggcgactg agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag
     5401 caatatttca agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaatctag
     5461 ctgcatcagg atcatatcgt cgggtctttt ttccggctca gtcatcgccc aagctggcgc
     5521 tatctgggca tcggggagga agaagcccgt gccttttccc gcgaggttga agcggcatgg
     5581 aaagagtttg ccgaggatga ctgctgctgc attgacgttg agcgaaaacg cacgtttacc
     5641 atgatgattc gggaaggtgt ggccatgcac gcctttaacg gtgaactgtt cgttcaggcc
     5701 acctgggata ccagttcgtc gcggcttttc cggacacagt tccggatggt cagcccgaag
     5761 cgcatcagca acccgaacaa taccggcgac agccggaact gccgtgccgg tgtgcagatt
     5821 aatgacagcg gtgcggcgct gggatattac gtcagcgagg acgggtatcc tggctggatg
     5881 ccgcagaaat ggacatggat accccgtgag ttacccggcg ggcgcgcttg gcgtaatcat
     5941 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     6001 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     6061 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6121 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     6181 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     6241 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6301 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6361 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6421 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6481 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6541 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6601 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6661 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6721 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6781 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6841 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6901 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6961 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     7021 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     7081 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     7141 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7201 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     7261 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7321 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7381 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7441 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7501 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7561 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7621 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7681 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7741 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7801 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7861 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7921 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7981 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     8041 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     8101 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     8161 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     8221 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     8281 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     8341 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     8401 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     8461 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     8521 cgcgcttaat gcgccgctac agggcgcgt
//
