LOCUS       sequence-297767-        8233 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid with 4xMxPylT C41CA 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 8233)
  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 8233)
  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_4xMx PylT C41CA_GFP 150TAG
FEATURES             Location/Qualifiers
     source          1..8233
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..63
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1538..1749
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1762..2030
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      2064..2073
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(2805..3379,3380..3382,3383..3391)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2972..2989)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3199..3218
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3404..3802
                     /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(3868..3888)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3930..4051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3967..3986)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4021..4040
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4814..4848
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5339..5355)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5339..5355)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5352..5374)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5363..5379
                     /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(5387..5393,5394..5411,5412..5417))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5432..5453
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5570..5587)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5741..6329)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5821..5840)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6500..7291,7292..7360))
                     /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     7123..7142
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7361..7465)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7491..7946)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7628..7649)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7840..7859
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8073..8095
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8087..8104
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8088..8104
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgtgta aaattgacgc
       61 atgtgtttta tcggtctgta tatcgaggtt tatttattaa tttgaataga tattaagttt
      121 tattatattt acacttacat actaataata aattcaacaa acaatttatt tatgtttatt
      181 tatttattaa aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa acttttatga
      241 gggacagccc ccccccaaag cccccaggga tgtaattacg tccctccccc gctagggggc
      301 agcagcgagc cgcccggggc tccgctccgg tccggcgctc cccccgcatc cccgagccgg
      361 cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg ggatcgcttt cctctgaacg
      421 cttctcgctg ctctttgagc ctgcagacac ctggggggat acggggaaaa ggcctccacg
      481 gccaactagt aaaaaccgca cttgtccgag agaccggggc gtcgaacccc gcttggctag
      541 gttttagaga cccgctggtc gccggaccgt cccccgaggt acccaggcgg cgcacaagct
      601 atataaacct gaaggaaatc tcaactttac acttaggtca agttacttat cgtactagag
      661 cttcagcagg aaatttaact aaaatctaat ttaaccagca tagcaaatat catttattcc
      721 caaaatgcta aagtttgaga taaacggact tgatttccgg ctgttttgac actatccaga
      781 atgccttgca gatgggtggg gcatgctaaa tactgcagca agaaaaaccg cacttgtccg
      841 agagaccggg gcgtcgaacc ccgcttggct aggttttaga gacccgctgg tcgccggacc
      901 gtcccccgag gtacccaggc ggcgcacaag ctatataaac ctgaaggaaa tctcaacttt
      961 acacttaggt caagttactt atcgtactag agcttcagca ggaaatttaa ctaaaatcta
     1021 atttaaccag catagcaaat atcatttatt cccaaaatgc taaagtttga gataaacgga
     1081 cttgatttcc ggctgttttg acactatcca gaatgccttg cagatgggtg gggcatgcta
     1141 aatactgcag cgataaaaac cgcactgtcc gagagaccgg ggcgtcgaac cccgcttggc
     1201 taggttttag agacccgctg gtcgccggac cgtcccccga ggtacccagg cggcgcacaa
     1261 gctatataaa cctgaaggaa atctcaactt tacacttagg tcaagttact tatcgtacta
     1321 gagcttcagc aggaaattta actaaaatct aatttaacca gcatagcaaa tatcatttat
     1381 tcccaaaatg ctaaagtttg agataaacgg acttgatttc cggctgtttt gacactatcc
     1441 agaatgcctt gcagatgggt ggggcatgct aaatactgca gctccaaggc cactagtgct
     1501 ctagaaagga tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
     1561 gtccccgaga agttgggggg aggggtcggc aattgaacgg gtgcctagag aaggtggcgc
     1621 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     1681 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     1741 agaacacagc tgaagcttcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc
     1801 tgaggccgcc atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc
     1861 tgaactgcgt ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg
     1921 gcgctccctt ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt
     1981 gctcaactct acgtctttgt ttcgttttct gttctgcgcc gttacagatc caagctgtga
     2041 ccggcgccta ctctagagct agcgccacca tggtgtccaa gggcgaggaa ctgttcaccg
     2101 gcgtggtgcc catcctggtg gaactggatg gcgacgtgaa cggccacaag ttctctgtgc
     2161 ggggagaggg cgaaggcgac gccacaaatg gcaagctgac cctggagttc atctgcacca
     2221 ccggcaagct gcccgtgcct tggcctaccc tcgtgaccac actgacctac ggcgtgcagt
     2281 gcttcagcag ataccccgac catatgaagc ggcacgactt cttcaagagc gccatgcccg
     2341 agggctacgt gcaggaacgg accatcagct tcaaggacga cggcacctac aagaccagag
     2401 ccgaagtgaa gttcgagggc gacaccctcg tgaaccggat cgagctgaag ggcatcgatt
     2461 tcaaagagga cggcaacatc ctgggccaca agctggagta caacttcaac agccactagg
     2521 tgtacatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag atccggcaca
     2581 acgtggaaga tggcagcgtg cagctggccg accactacca gcagaacacc cccatcggag
     2641 atggccccgt gctgctgccc gacaaccact acctgagcac ccagagcgtg ctgagcaagg
     2701 accccaacga gaagcgggac cacatggtgc tgctggaatt cgtgaccgcc gctggcatca
     2761 cccacggcat ggacgagctg tacaagggca gctgagcggc cgcgcccctc tccctccccc
     2821 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
     2881 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
     2941 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg
     3001 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     3061 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     3121 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     3181 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     3241 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt
     3301 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
     3361 tgaaaaacac gatgataata tggccacaac catggcgtcc ggaatggcca agcctttgtc
     3421 tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc
     3481 tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt
     3541 caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc
     3601 tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat
     3661 cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc
     3721 catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc
     3781 tggttatgtg tgggagggct gaccatgggt cgacaatcaa cctctggatt acaaaatttg
     3841 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     3901 tttaatgcct ttgtatcatg cgttaactaa acttgtttat tgcagcttat aatggttaca
     3961 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4021 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gaattgactc aaatgatgtc
     4081 aattagtcta tcagaagctc atctggtctc ccttccgggg gacaagacat ccctgtttaa
     4141 tatttaaaca gcagtgttcc caaactgggt tcttatatcc cttgctctgg tcaaccaggt
     4201 tgcagggttt cctgtcctca caggaacgaa gtccctaaag aaacagtggc agccaggttt
     4261 agccccggaa ttgactggat tcctttttta gggcccattg gtatggcttt ttccccgtat
     4321 ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg
     4381 tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga
     4441 gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg
     4501 taattacatc cctgggggct ttgggggggg gctgtccctg atatctataa caagaaaata
     4561 tatatataat aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag
     4621 ttaaatctta aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta
     4681 tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg
     4741 gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa
     4801 tatttcaaga atgcatgcgt caattttacg cagactatct ttctagggtt aatctagctg
     4861 catcaggatc atatcgtcgg gtcttttttc cggctcagtc atcgcccaag ctggcgctat
     4921 ctgggcatcg gggaggaaga agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa
     4981 gagtttgccg aggatgactg ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg
     5041 atgattcggg aaggtgtggc catgcacgcc tttaacggtg aactgttcgt tcaggccacc
     5101 tgggatacca gttcgtcgcg gcttttccgg acacagttcc ggatggtcag cccgaagcgc
     5161 atcagcaacc cgaacaatac cggcgacagc cggaactgcc gtgccggtgt gcagattaat
     5221 gacagcggtg cggcgctggg atattacgtc agcgaggacg ggtatcctgg ctggatgccg
     5281 cagaaatgga catggatacc ccgtgagtta cccggcgggc gcgcttggcg taatcatggt
     5341 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5401 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5461 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5521 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5581 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5641 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5701 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5761 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5821 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5881 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5941 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6001 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6061 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6121 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6181 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6241 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6301 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6361 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6421 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6481 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6541 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6601 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6661 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6721 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6781 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6841 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6901 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6961 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     7021 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7081 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7141 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7201 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7261 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7321 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7381 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7441 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     7501 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     7561 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     7621 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     7681 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     7741 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     7801 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     7861 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     7921 gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga
     7981 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     8041 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     8101 cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg gaggacgggc agactcgcgg
     8161 tgcaaatgtg ttttacagcg tgatggagca gatgaagatg ctcgacacgc tgcagaacac
     8221 gcagctagat taa
//
