LOCUS       sequence-297733-        8562 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid with 4xMxPylT 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 8562)
  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 8562)
  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_GFP 150TAG
FEATURES             Location/Qualifiers
     source          1..8562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(91..546)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(228..249)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     440..459
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     673..695
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     687..704
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     688..704
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   834..896
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2700..2911
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2924..3192
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      3226..3235
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(3967..4541,4542..4544,4545..4553)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4134..4151)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4361..4380
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4566..4964
                     /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(5030..5050)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5092..5213
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5129..5148)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5183..5202
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5976..6010
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6501..6517)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6501..6517)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6514..6536)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6525..6541
                     /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(6549..6555,6556..6573,6574..6579))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6594..6615
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6732..6749)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6903..7491)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6983..7002)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7662..8453,8454..8522))
                     /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     8285..8304
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8523..8562,1..65))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
       61 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
      121 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
      181 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
      241 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
      301 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
      361 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
      421 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
      481 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
      541 gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
      601 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
      661 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcctcgttca
      721 ttcacgtttt tgaacccgtg gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg
      781 tgatggagca gatgaagatg ctcgacacgc tgcagaacac gcagctagat taaccctaga
      841 aagataatca tattgtgacg tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt
      901 ttatcggtct gtatatcgag gtttatttat taatttgaat agatattaag ttttattata
      961 tttacactta catactaata ataaattcaa caaacaattt atttatgttt atttatttat
     1021 taaaaaaaac aaaaactcaa aatttcttct ataaagtaac aaaactttta tgagggacag
     1081 ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg
     1141 agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg
     1201 cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg
     1261 ctgctctttg agcctgcaga cacctggggg gatacgggga aaaggcctcc acggccaact
     1321 agtaaaaacc gcacttgtcc gagagaccgg ggcgtcgaac cccgctggct aggttttaga
     1381 gacccgctgg tcgccggacc gtcccccgag gtacccaggc ggcgcacaag ctatataaac
     1441 ctgaaggaaa tctcaacttt acacttaggt caagttactt atcgtactag agcttcagca
     1501 ggaaatttaa ctaaaatcta atttaaccag catagcaaat atcatttatt cccaaaatgc
     1561 taaagtttga gataaacgga cttgatttcc ggctgttttg acactatcca gaatgccttg
     1621 cagatgggtg gggcatgcta aatactgcag gcaaaaaaac cgcacttgtc cgagagaccg
     1681 gggcgtcgaa ccccgctggc taggttttag agacccgctg gtcgccggac cgtcccccga
     1741 ggtacccagg cggcgcacaa gctatataaa cctgaaggaa atctcaactt tacacttagg
     1801 tcaagttact tatcgtacta gagcttcagc aggaaattta actaaaatct aatttaacca
     1861 gcatagcaaa tatcatttat tcccaaaatg ctaaagtttg agataaacgg acttgatttc
     1921 cggctgtttt gacactatcc agaatgcctt gcagatgggt ggggcatgct aaatactgca
     1981 gcaagaaaaa ccgcacttgt ccgagagacc ggggcgtcga accccgctgg ctaggtttta
     2041 gagacccgct ggtcgccgga ccgtcccccg aggtacccag gcggcgcaca agctatataa
     2101 acctgaagga aatctcaact ttacacttag gtcaagttac ttatcgtact agagcttcag
     2161 caggaaattt aactaaaatc taatttaacc agcatagcaa atatcattta ttcccaaaat
     2221 gctaaagttt gagataaacg gacttgattt ccggctgttt tgacactatc cagaatgcct
     2281 tgcagatggg tggggcatgc taaatactgc agcgataaaa accgcacttg tccgagagac
     2341 cggggcgtcg aaccccgctg gctaggtttt agagacccgc tggtcgccgg accgtccccc
     2401 gaggtaccca ggcggcgcac aagctatata aacctgaagg aaatctcaac tttacactta
     2461 ggtcaagtta cttatcgtac tagagcttca gcaggaaatt taactaaaat ctaatttaac
     2521 cagcatagca aatatcattt attcccaaaa tgctaaagtt tgagataaac ggacttgatt
     2581 tccggctgtt ttgacactat ccagaatgcc ttgcagatgg gtggggcatg ctaaatactg
     2641 cagctccaag gccactagtg ctctagaaag gatctgcgat cgctccggtg cccgtcagtg
     2701 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
     2761 gggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
     2821 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
     2881 ttttcgcaac gggtttgccg ccagaacaca gctgaagctt cgaggggctc gcatctctcc
     2941 ttcacgcgcc cgccgcccta cctgaggccg ccatccacgc cggttgagtc gcgttctgcc
     3001 gcctcccgcc tgtggtgcct cctgaactgc gtccgccgtc taggtaagtt taaagctcag
     3061 gtcgagaccg ggcctttgtc cggcgctccc ttggagccta cctagactca gccggctctc
     3121 cacgctttgc ctgaccctgc ttgctcaact ctacgtcttt gtttcgtttt ctgttctgcg
     3181 ccgttacaga tccaagctgt gaccggcgcc tactctagag ctagcgccac catggtgtcc
     3241 aagggcgagg aactgttcac cggcgtggtg cccatcctgg tggaactgga tggcgacgtg
     3301 aacggccaca agttctctgt gcggggagag ggcgaaggcg acgccacaaa tggcaagctg
     3361 accctggagt tcatctgcac caccggcaag ctgcccgtgc cttggcctac cctcgtgacc
     3421 acactgacct acggcgtgca gtgcttcagc agataccccg accatatgaa gcggcacgac
     3481 ttcttcaaga gcgccatgcc cgagggctac gtgcaggaac ggaccatcag cttcaaggac
     3541 gacggcacct acaagaccag agccgaagtg aagttcgagg gcgacaccct cgtgaaccgg
     3601 atcgagctga agggcatcga tttcaaagag gacggcaaca tcctgggcca caagctggag
     3661 tacaacttca acagccacta ggtgtacatc accgccgaca agcagaagaa cggcatcaag
     3721 gccaacttca agatccggca caacgtggaa gatggcagcg tgcagctggc cgaccactac
     3781 cagcagaaca cccccatcgg agatggcccc gtgctgctgc ccgacaacca ctacctgagc
     3841 acccagagcg tgctgagcaa ggaccccaac gagaagcggg accacatggt gctgctggaa
     3901 ttcgtgaccg ccgctggcat cacccacggc atggacgagc tgtacaaggg cagctgagcg
     3961 gccgcgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg
     4021 ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag
     4081 ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc
     4141 caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg
     4201 aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag
     4261 gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca
     4321 gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt
     4381 caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc
     4441 tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa
     4501 ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca accatggcgt
     4561 ccggaatggc caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta
     4621 caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg
     4681 acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac
     4741 tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga
     4801 tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg
     4861 atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg
     4921 ggattcgtga attgctgccc tctggttatg tgtgggaggg ctgaccatgg gtcgacaatc
     4981 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     5041 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgcgttaact aaacttgttt
     5101 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     5161 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     5221 tggaattgac tcaaatgatg tcaattagtc tatcagaagc tcatctggtc tcccttccgg
     5281 gggacaagac atccctgttt aatatttaaa cagcagtgtt cccaaactgg gttcttatat
     5341 cccttgctct ggtcaaccag gttgcagggt ttcctgtcct cacaggaacg aagtccctaa
     5401 agaaacagtg gcagccaggt ttagccccgg aattgactgg attccttttt tagggcccat
     5461 tggtatggct ttttccccgt atccccccag gtgtctgcag gctcaaagag cagcgagaag
     5521 cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg
     5581 ccggctcggg gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct
     5641 gccccctagc gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc
     5701 tgatatctat aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat
     5761 aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc
     5821 attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca
     5881 cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc
     5941 gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat
     6001 ctttctaggg ttaatctagc tgcatcagga tcatatcgtc gggtcttttt tccggctcag
     6061 tcatcgccca agctggcgct atctgggcat cggggaggaa gaagcccgtg ccttttcccg
     6121 cgaggttgaa gcggcatgga aagagtttgc cgaggatgac tgctgctgca ttgacgttga
     6181 gcgaaaacgc acgtttacca tgatgattcg ggaaggtgtg gccatgcacg cctttaacgg
     6241 tgaactgttc gttcaggcca cctgggatac cagttcgtcg cggcttttcc ggacacagtt
     6301 ccggatggtc agcccgaagc gcatcagcaa cccgaacaat accggcgaca gccggaactg
     6361 ccgtgccggt gtgcagatta atgacagcgg tgcggcgctg ggatattacg tcagcgagga
     6421 cgggtatcct ggctggatgc cgcagaaatg gacatggata ccccgtgagt tacccggcgg
     6481 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     6541 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     6601 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     6661 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     6721 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     6781 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     6841 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6901 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6961 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     7021 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     7081 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     7141 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     7201 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     7261 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     7321 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     7381 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     7441 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     7501 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     7561 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     7621 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     7681 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     7741 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     7801 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7861 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7921 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7981 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     8041 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     8101 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     8161 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     8221 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     8281 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     8341 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     8401 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     8461 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     8521 atactcttcc tttttcaata ttattgaagc atttatcagg gt
//
