LOCUS       sequence-297667-        8565 bp    DNA     circular SYN 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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8565)
  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 8565)
  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..8565
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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        2721..2932
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2945..3213
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      3247..3256
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3976..3993
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    join(4010..4584,4585..4587,4588..4596)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4177..4194)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4404..4423
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4609..5007
                     /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(5073..5093)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5135..5256
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5172..5191)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5226..5245
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6019..6053
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6544..6560)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6544..6560)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6557..6579)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6568..6584
                     /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(6592..6598,6599..6616,6617..6622))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6637..6658
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6775..6792)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6946..7534)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7026..7045)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7705..8496,8497..8565))
                     /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     8328..8347
                     /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 aggamaarnn nnnnggmtac cccgggaatc taacccggct gaacggattt
     1741 agagtccgtt cgatctacat gatcaggttt ccgaggtacc caggcggcgc acaagctata
     1801 taaacctgaa ggaaatctca actttacact taggtcaagt tacttatcgt actagagctt
     1861 cagcaggaaa tttaactaaa atctaattta accagcatag caaatatcat ttattcccaa
     1921 aatgctaaag tttgagataa acggacttga tttccggctg ttttgacact atccagaatg
     1981 ccttgcagat gggtggggca tgctaaatac tgcaggaaaa accgcacttg tccggaaacc
     2041 ccgggaatct aacccggctg aacggattta gagtccgttc gatctacatg atcaggtttc
     2101 cgaggtaccc aggcggcgca caagctatat aaacctgaag gaaatctcaa ctttacactt
     2161 aggtcaagtt acttatcgta ctagagcttc agcaggaaat ttaactaaaa tctaatttaa
     2221 ccagcatagc aaatatcatt tattcccaaa atgctaaagt ttgagataaa cggacttgat
     2281 ttccggctgt tttgacacta tccagaatgc cttgcagatg ggtggggcat gctaaatact
     2341 gcaggaaaaa ccgcacttgt ccggaaaccc cgggaatcta acccggctga acggatttag
     2401 agtccgttcg atctacatga tcaggtttcc gaggtaccca ggcggcgcac aagctatata
     2461 aacctgaagg aaatctcaac tttacactta ggtcaagtta cttatcgtac tagagcttca
     2521 gcaggaaatt taactaaaat ctaatttaac cagcatagca aatatcattt attcccaaaa
     2581 tgctaaagtt tgagataaac ggacttgatt tccggctgtt ttgacactat ccagaatgcc
     2641 ttgcagatgg gtggggcatg ctaaatactg cagaactagt gctctagaaa ggatctgcga
     2701 tcgctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
     2761 gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa actgggaaag
     2821 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
     2881 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac agctgaagct
     2941 tcgaggggct cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg
     3001 ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt
     3061 ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct
     3121 acctagactc agccggctct ccacgctttg cctgaccctg cttgctcaac tctacgtctt
     3181 tgtttcgttt tctgttctgc gccgttacag atccaagctg tgaccggcgc ctactctaga
     3241 gctagcgcca ccatggtgtc caagggcgag gaactgttca ccggcgtggt gcccatcctg
     3301 gtggaactgg atggcgacgt gaacggccac aagttctctg tgcggggaga gggcgaaggc
     3361 gacgccacaa atggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     3421 ccttggccta ccctcgtgac cacactgacc tacggcgtgc agtgcttcag cagatacccc
     3481 gaccatatga agcggcacga cttcttcaag agcgccatgc ccgagggcta cgtgcaggaa
     3541 cggaccatca gcttcaagga cgacggcacc tacaagacca gagccgaagt gaagttcgag
     3601 ggcgacaccc tcgtgaaccg gatcgagctg aagggcatcg atttcaaaga ggacggcaac
     3661 atcctgggcc acaagctgga gtacaacttc aacagccact aggtgtacat caccgccgac
     3721 aagcagaaga acggcatcaa ggccaacttc aagatccggc acaacgtgga agatggcagc
     3781 gtgcagctgg ccgaccacta ccagcagaac acccccatcg gagatggccc cgtgctgctg
     3841 cccgacaacc actacctgag cacccagagc gtgctgagca aggaccccaa cgagaagcgg
     3901 gaccacatgg tgctgctgga attcgtgacc gccgctggca tcacccacgg catggacgag
     3961 ctgtacaagg gcagccacca tcaccatcac cattaataat aaggccgcgc ccctctccct
     4021 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
     4081 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
     4141 ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
     4201 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg
     4261 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
     4321 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
     4381 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
     4441 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta
     4501 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     4561 tcctttgaaa aacacgatga taatatggcc acaaccatgg cgtccggaat ggccaagcct
     4621 ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa cagcatcccc
     4681 atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg catcttcact
     4741 ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt gctgggcact
     4801 gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa tgagaacagg
     4861 ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca tcctgggatc
     4921 aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg
     4981 ccctctggtt atgtgtggga gggctgacca tgggtcgaca atcaacctct ggattacaaa
     5041 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     5101 gctgctttaa tgcctttgta tcatgcgtta actaaacttg tttattgcag cttataatgg
     5161 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     5221 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggaatt gactcaaatg
     5281 atgtcaatta gtctatcaga agctcatctg gtctcccttc cgggggacaa gacatccctg
     5341 tttaatattt aaacagcagt gttcccaaac tgggttctta tatcccttgc tctggtcaac
     5401 caggttgcag ggtttcctgt cctcacagga acgaagtccc taaagaaaca gtggcagcca
     5461 ggtttagccc cggaattgac tggattcctt ttttagggcc cattggtatg gctttttccc
     5521 cgtatccccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga
     5581 tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg
     5641 ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag
     5701 ggacgtaatt acatccctgg gggctttggg ggggggctgt ccctgatatc tataacaaga
     5761 aaatatatat ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt
     5821 atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt
     5881 cgttatagtt caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc
     5941 aagcggcgac tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag
     6001 agcaatattt caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaatct
     6061 agctgcatca ggatcatatc gtcgggtctt ttttccggct cagtcatcgc ccaagctggc
     6121 gctatctggg catcggggag gaagaagccc gtgccttttc ccgcgaggtt gaagcggcat
     6181 ggaaagagtt tgccgaggat gactgctgct gcattgacgt tgagcgaaaa cgcacgttta
     6241 ccatgatgat tcgggaaggt gtggccatgc acgcctttaa cggtgaactg ttcgttcagg
     6301 ccacctggga taccagttcg tcgcggcttt tccggacaca gttccggatg gtcagcccga
     6361 agcgcatcag caacccgaac aataccggcg acagccggaa ctgccgtgcc ggtgtgcaga
     6421 ttaatgacag cggtgcggcg ctgggatatt acgtcagcga ggacgggtat cctggctgga
     6481 tgccgcagaa atggacatgg ataccccgtg agttacccgg cgggcgcgct tggcgtaatc
     6541 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     6601 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     6661 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     6721 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     6781 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     6841 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     6901 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     6961 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     7021 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     7081 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     7141 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     7201 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     7261 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     7321 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     7381 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     7441 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     7501 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     7561 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     7621 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     7681 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     7741 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7801 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     7861 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     7921 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     7981 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     8041 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     8101 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     8161 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     8221 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     8281 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     8341 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     8401 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     8461 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     8521 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcat
//
