LOCUS       sequence-296092-        8157 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid with 4xMmaPylT cassette to receive genes of interest for 
            amber suppression; for transient or stable piggyBac-mediated 
            integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8157)
  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 8157)
  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
FEATURES             Location/Qualifiers
     source          1..8157
                     /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        2687..2898
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2911..3179
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     intron          3282..3511
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     3494..3513
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(3562..4136,4137..4139,4140..4148)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3729..3746)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3956..3975
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4161..4559
                     /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(4625..4645)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4687..4808
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4724..4743)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4778..4797
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5571..5605
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6096..6112)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6096..6112)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6109..6131)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6120..6136
                     /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(6144..6150,6151..6168,6169..6174))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6189..6210
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6327..6344)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6498..7086)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6578..6597)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7257..8048,8049..8117))
                     /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     7880..7899
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8118..8157,1..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 agtgaaaaac cgcacttgtc cggaaacccc gggaatctaa cccggctgaa cggatttaga
     1381 gtccgttcga tctacatgat caggtttccg aggtacccag gcggcgcaca agctatataa
     1441 acctgaagga aatctcaact ttacacttag gtcaagttac ttatcgtact agagcttcag
     1501 caggaaattt aactaaaatc taatttaacc agcatagcaa atatcattta ttcccaaaat
     1561 gctaaagttt gagataaacg gacttgattt ccggctgttt tgacactatc cagaatgcct
     1621 tgcagatggg tggggcatgc taaatactgc aggaaaaacc gcacttgtcc ggaaaccccg
     1681 ggaatctaac ccggctgaac ggatttagag tccgttcgat ctacatgatc aggtttccga
     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 ggaaaaaccg cacttgtccg gaaaccccgg gaatctaacc cggctgaacg gatttagagt
     2041 ccgttcgatc tacatgatca ggtttccgag gtacccaggc ggcgcacaag ctatataaac
     2101 ctgaaggaaa tctcaacttt acacttaggt caagttactt atcgtactag agcttcagca
     2161 ggaaatttaa ctaaaatcta atttaaccag catagcaaat atcatttatt cccaaaatgc
     2221 taaagtttga gataaacgga cttgatttcc ggctgttttg acactatcca gaatgccttg
     2281 cagatgggtg gggcatgcta aatactgcag gaaaaaccgc acttgtccgg aaaccccggg
     2341 aatctaaccc ggctgaacgg atttagagtc cgttcgatct acatgatcag gtttccgagg
     2401 tacccaggcg gcgcacaagc tatataaacc tgaaggaaat ctcaacttta cacttaggtc
     2461 aagttactta tcgtactaga gcttcagcag gaaatttaac taaaatctaa tttaaccagc
     2521 atagcaaata tcatttattc ccaaaatgct aaagtttgag ataaacggac ttgatttccg
     2581 gctgttttga cactatccag aatgccttgc agatgggtgg ggcatgctaa atactgcaga
     2641 actagtgctc tagaaaggat ctgcgatcgc tccggtgccc gtcagtgggc agagcgcaca
     2701 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg tgcctagaga
     2761 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     2821 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     2881 tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc acgcgcccgc
     2941 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt
     3001 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc
     3061 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg
     3121 accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg ttacagatcc
     3181 aagctgtgac cggcgcctac tctagagcta gcgaattcga atttaaatcg gatccgcggc
     3241 cgctggaatt aattcgctgt ctgcgagggc cagctgttgg ggtgagtact ccctctcaaa
     3301 agcgggcatg acttctgcgc taagattgtc agtttccaaa aacgaggagg atttgatatt
     3361 cacctggccc gcggtgatgc ctttgagggt ggccgcgtcc atctggtcag aaaagacaat
     3421 ctttttgttg tcaagcttga ggtgtggcag gcttgagatc tggccataca cttgagtgac
     3481 aatgacatcc actttgcctt tctctccaca ggtgtccact cccaggtcca actgcaggtc
     3541 gagcatgctc gagcggccgc gcccctctcc ctcccccccc cctaacgtta ctggccgaag
     3601 ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc
     3661 ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg
     3721 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc
     3781 tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc
     3841 cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa
     3901 ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct
     3961 ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg
     4021 atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg
     4081 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg
     4141 ccacaaccat ggcgtccgga atggccaagc ctttgtctca agaagaatcc accctcattg
     4201 aaagagcaac ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg
     4261 cagctctctc tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg
     4321 gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga
     4381 cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc
     4441 gacaggtgct tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac
     4501 agccgacggc agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctgac
     4561 catgggtcga caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     4621 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcgt
     4681 taactaaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4741 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4801 gtatcttatc atgtctggaa ttgactcaaa tgatgtcaat tagtctatca gaagctcatc
     4861 tggtctccct tccgggggac aagacatccc tgtttaatat ttaaacagca gtgttcccaa
     4921 actgggttct tatatccctt gctctggtca accaggttgc agggtttcct gtcctcacag
     4981 gaacgaagtc cctaaagaaa cagtggcagc caggtttagc cccggaattg actggattcc
     5041 ttttttaggg cccattggta tggctttttc cccgtatccc cccaggtgtc tgcaggctca
     5101 aagagcagcg agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc
     5161 tgtccccgca cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc
     5221 gggcggctcg ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg
     5281 ggggggggct gtccctgata tctataacaa gaaaatatat atataataag ttatcacgta
     5341 agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa
     5401 agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta
     5461 ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca
     5521 cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa
     5581 ttttacgcag actatctttc tagggttaat ctagctgcat caggatcata tcgtcgggtc
     5641 ttttttccgg ctcagtcatc gcccaagctg gcgctatctg ggcatcgggg aggaagaagc
     5701 ccgtgccttt tcccgcgagg ttgaagcggc atggaaagag tttgccgagg atgactgctg
     5761 ctgcattgac gttgagcgaa aacgcacgtt taccatgatg attcgggaag gtgtggccat
     5821 gcacgccttt aacggtgaac tgttcgttca ggccacctgg gataccagtt cgtcgcggct
     5881 tttccggaca cagttccgga tggtcagccc gaagcgcatc agcaacccga acaataccgg
     5941 cgacagccgg aactgccgtg ccggtgtgca gattaatgac agcggtgcgg cgctgggata
     6001 ttacgtcagc gaggacgggt atcctggctg gatgccgcag aaatggacat ggataccccg
     6061 tgagttaccc ggcgggcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     6121 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     6181 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     6241 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     6301 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     6361 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     6421 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     6481 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     6541 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     6601 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     6661 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6721 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6781 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6841 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6901 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     6961 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     7021 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     7081 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     7141 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     7201 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     7261 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     7321 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     7381 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     7441 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     7501 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     7561 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     7621 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     7681 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     7741 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7801 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7861 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7921 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7981 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     8041 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     8101 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggt
//
