LOCUS       sequence-309610-        7744 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid with Mx1201 PylRS for amber suppression in transient or 
            stable, piggybac-mediated, integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7744)
  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 7744)
  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_FLAG-Mx1201 PylRS
FEATURES             Location/Qualifiers
     source          1..7744
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     117..139
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     131..148
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     132..148
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   278..340
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        808..1019
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1032..1300
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             1348..1371
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2221..2793
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2388..2405)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2615..2634
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2802..3596
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLAATGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2856..2875)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3466..3485
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3656..3676)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3718..3839
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3755..3774)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3809..3828
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4602..4636
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5127..5143)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5127..5143)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5140..5162)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5151..5167
                     /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(5175..5181,5182..5199,5200..5205))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5220..5241
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5358..5375)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5529..6117)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5609..5628)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6288..7079,7080..7148))
                     /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     6911..6930
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7149..7253)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7279..7734)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7416..7437)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7628..7647
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
       61 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
      121 gtcacgacgt tgtaaaacga cggccagtga gcgcgcctcg ttcattcacg tttttgaacc
      181 cgtggaggac gggcagactc gcggtgcaaa tgtgttttac agcgtgatgg agcagatgaa
      241 gatgctcgac acgctgcaga acacgcagct agattaaccc tagaaagata atcatattgt
      301 gacgtacgtt aaagataatc atgtgtaaaa ttgacgcatg tgttttatcg gtctgtatat
      361 cgaggtttat ttattaattt gaatagatat taagttttat tatatttaca cttacatact
      421 aataataaat tcaacaaaca atttatttat gtttatttat ttattaaaaa aaacaaaaac
      481 tcaaaatttc ttctataaag taacaaaact tttatgaggg acagcccccc cccaaagccc
      541 ccagggatgt aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc
      601 gctccggtcc ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc
      661 acggggaagg tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg
      721 cagacacctg gggggatacg gggaaaaggc ctccacggcc aactagtgct ctagaaagga
      781 tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga
      841 agttgggggg aggggtcggc aattgaacgg gtgcctagag aaggtggcgc ggggtaaact
      901 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata
      961 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc
     1021 tgaagcttcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgcc
     1081 atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt
     1141 ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt
     1201 ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct
     1261 acgtctttgt ttcgttttct gttctgcgcc gttacagatc caagctgtga ccggcgccta
     1321 ctctagagct agctcgcgac caccatggac tacaaggacg acgacgacaa gacggtaaag
     1381 tatacggatg cacagataca gcgccttcgc gaatacggca acgggaccta tgagcagaag
     1441 gtcttcgagg acctcgcatc gagggatgct gccttctcca aggagatgtc cgtcgcctct
     1501 accgacaacg agaagaagat caaggggatg atcgccaatc cgtcccgtca tggattgacc
     1561 cagctgatga acgacatcgc cgacgcattg gtcgccgagg gtttcatcga ggtccgtacg
     1621 cccatattca tatcgaagga tgcgctggca cgtatgacca tcaccgagga caagcccctt
     1681 ttcaagcagg tcttctggat cgacgagaaa agggcgctca ggcctatgct ggcacctaac
     1741 ctttattccg tcatgaggga cctgagggac catacggacg gtccggtgaa gatcttcgag
     1801 atgggttcct gcttcaggaa ggagtcccac agcgggatgc atctggagga gttcaccatg
     1861 ctgaacctcg tggacatggg tccccgcgga gacgccacgg aggtcctgaa gaactacata
     1921 tcggtcgtga tgaaggcggc cggtctcccg gactacgacc tcgtacagga ggagtccgac
     1981 gtatacaagg agaccataga cgtggagatc aacggtcagg aggtctgttc cgcagccgtc
     2041 ggtccacact atctcgatgc ggcccacgat gtccacgagc cttggtccgg agcgggattc
     2101 ggtctcgaac gcctgctgac catcagggag aagtacagca ccgtgaagaa gggaggagcc
     2161 agcatcagct acctcaacgg tgcgaagatc aactgatgaa tcgatggatc cgcggccgcg
     2221 cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg
     2281 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg
     2341 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg
     2401 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca
     2461 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct
     2521 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca
     2581 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa
     2641 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg
     2701 cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg
     2761 ggacgtggtt ttcctttgaa aaacacgata atacctccgg aatgattgaa caagatggat
     2821 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     2881 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     2941 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     3001 tatcgtggct ggccgcgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     3061 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     3121 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     3181 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     3241 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     3301 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     3361 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     3421 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     3481 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     3541 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagtcg
     3601 acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     3661 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgcg ttaactaaac
     3721 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3781 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     3841 catgtctgga attgactcaa atgatgtcaa ttagtctatc agaagctcat ctggtctccc
     3901 ttccggggga caagacatcc ctgtttaata tttaaacagc agtgttccca aactgggttc
     3961 ttatatccct tgctctggtc aaccaggttg cagggtttcc tgtcctcaca ggaacgaagt
     4021 ccctaaagaa acagtggcag ccaggtttag ccccggaatt gactggattc cttttttagg
     4081 gcccattggt atggcttttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc
     4141 gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc
     4201 acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc
     4261 gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc
     4321 tgtccctgat atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca
     4381 tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca
     4441 tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga
     4501 caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg
     4561 attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca
     4621 gactatcttt ctagggttaa tctagctgca tcaggatcat atcgtcgggt cttttttccg
     4681 gctcagtcat cgcccaagct ggcgctatct gggcatcggg gaggaagaag cccgtgcctt
     4741 ttcccgcgag gttgaagcgg catggaaaga gtttgccgag gatgactgct gctgcattga
     4801 cgttgagcga aaacgcacgt ttaccatgat gattcgggaa ggtgtggcca tgcacgcctt
     4861 taacggtgaa ctgttcgttc aggccacctg ggataccagt tcgtcgcggc ttttccggac
     4921 acagttccgg atggtcagcc cgaagcgcat cagcaacccg aacaataccg gcgacagccg
     4981 gaactgccgt gccggtgtgc agattaatga cagcggtgcg gcgctgggat attacgtcag
     5041 cgaggacggg tatcctggct ggatgccgca gaaatggaca tggatacccc gtgagttacc
     5101 cggcgggcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5161 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5221 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5281 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5341 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5401 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5461 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5521 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5581 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5641 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5701 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5761 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5821 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5881 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5941 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     6001 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6061 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6121 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6181 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6241 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6301 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6361 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6421 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6481 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6541 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6601 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6661 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6721 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6781 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6841 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6901 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6961 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7021 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7081 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7141 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7201 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7261 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     7321 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     7381 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     7441 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     7501 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     7561 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     7621 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     7681 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     7741 cgcc
//
