LOCUS       sequence-297753-        8752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid with 4xMx PylT C41CA cassette and MX1201 PylRS 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 8752)
  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 8752)
  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_FLAG_Mx PylRS
FEATURES             Location/Qualifiers
     source          1..8752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1309..1520
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1533..1801
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             1849..1872
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2722..3294
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2889..2906)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3116..3135
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3303..4097
                     /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(3357..3376)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3967..3986
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4157..4177)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4219..4340
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4256..4275)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4310..4329
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5103..5137
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5628..5644)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5628..5644)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5641..5663)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5652..5668
                     /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(5676..5682,5683..5700,5701..5706))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5721..5742
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5859..5876)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6030..6618)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6110..6129)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6789..7580,7581..7649))
                     /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     7412..7431
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7650..7754)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7780..8235)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7917..7938)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8129..8148
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8362..8384
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8376..8393
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8377..8393
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   8523..8585
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 acttttatga gggacagccc ccccccaaag cccccaggga tgtaattacg tccctccccc
       61 gctagggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc cccccgcatc
      121 cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg ggatcgcttt
      181 cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat acggggaaaa
      241 ggcctccacg gccaactagt aaaaaccgca cttgtccgag agaccggggc gtcgaacccc
      301 gcttggctag gttttagaga cccgctggtc gccggaccgt cccccgaggt acccaggcgg
      361 cgcacaagct atataaacct gaaggaaatc tcaactttac acttaggtca agttacttat
      421 cgtactagag cttcagcagg aaatttaact aaaatctaat ttaaccagca tagcaaatat
      481 catttattcc caaaatgcta aagtttgaga taaacggact tgatttccgg ctgttttgac
      541 actatccaga atgccttgca gatgggtggg gcatgctaaa tactgcagca agaaaaaccg
      601 cacttgtccg agagaccggg gcgtcgaacc ccgcttggct aggttttaga gacccgctgg
      661 tcgccggacc gtcccccgag gtacccaggc ggcgcacaag ctatataaac ctgaaggaaa
      721 tctcaacttt acacttaggt caagttactt atcgtactag agcttcagca ggaaatttaa
      781 ctaaaatcta atttaaccag catagcaaat atcatttatt cccaaaatgc taaagtttga
      841 gataaacgga cttgatttcc ggctgttttg acactatcca gaatgccttg cagatgggtg
      901 gggcatgcta aatactgcag cgataaaaac cgcacttgtc cgagagaccg gggcgtcgaa
      961 ccccgcttgg ctaggtttta gagacccgct ggtcgccgga ccgtcccccg aggtacccag
     1021 gcggcgcaca agctatataa acctgaagga aatctcaact ttacacttag gtcaagttac
     1081 ttatcgtact agagcttcag caggaaattt aactaaaatc taatttaacc agcatagcaa
     1141 atatcattta ttcccaaaat gctaaagttt gagataaacg gacttgattt ccggctgttt
     1201 tgacactatc cagaatgcct tgcagatggg tggggcatgc taaatactgc agctccaagg
     1261 ccactagtgc tctagaaagg atctgcgatc gctccggtgc ccgtcagtgg gcagagcgca
     1321 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacg ggtgcctaga
     1381 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
     1441 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     1501 ggtttgccgc cagaacacag ctgaagcttc gaggggctcg catctctcct tcacgcgccc
     1561 gccgccctac ctgaggccgc catccacgcc ggttgagtcg cgttctgccg cctcccgcct
     1621 gtggtgcctc ctgaactgcg tccgccgtct aggtaagttt aaagctcagg tcgagaccgg
     1681 gcctttgtcc ggcgctccct tggagcctac ctagactcag ccggctctcc acgctttgcc
     1741 tgaccctgct tgctcaactc tacgtctttg tttcgttttc tgttctgcgc cgttacagat
     1801 ccaagctgtg accggcgcct actctagagc tagctcgcga ccaccatgga ctacaaggac
     1861 gacgacgaca agacggtaaa gtatacggat gcacagatac agcgccttcg cgaatacggc
     1921 aacgggacct atgagcagaa ggtcttcgag gacctcgcat cgagggatgc tgccttctcc
     1981 aaggagatgt ccgtcgcctc taccgacaac gagaagaaga tcaaggggat gatcgccaat
     2041 ccgtcccgtc atggattgac ccagctgatg aacgacatcg ccgacgcatt ggtcgccgag
     2101 ggtttcatcg aggtccgtac gcccatattc atatcgaagg atgcgctggc acgtatgacc
     2161 atcaccgagg acaagcccct tttcaagcag gtcttctgga tcgacgagaa aagggcgctc
     2221 aggcctatgc tggcacctaa cctttattcc gtcatgaggg acctgaggga ccatacggac
     2281 ggtccggtga agatcttcga gatgggttcc tgcttcagga aggagtccca cagcgggatg
     2341 catctggagg agttcaccat gctgaacctc gtggacatgg gtccccgcgg agacgccacg
     2401 gaggtcctga agaactacat atcggtcgtg atgaaggcgg ccggtctccc ggactacgac
     2461 ctcgtacagg aggagtccga cgtatacaag gagaccatag acgtggagat caacggtcag
     2521 gaggtctgtt ccgcagccgt cggtccacac tatctcgatg cggcccacga tgtccacgag
     2581 ccttggtccg gagcgggatt cggtctcgaa cgcctgctga ccatcaggga gaagtacagc
     2641 accgtgaaga agggaggagc cagcatcagc tacctcaacg gtgcgaagat caactgatga
     2701 atcgatggat ccgcggccgc gcccctctcc ctcccccccc cctaacgtta ctggccgaag
     2761 ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc
     2821 ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg
     2881 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc
     2941 tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc
     3001 cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa
     3061 ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct
     3121 ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg
     3181 atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg
     3241 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat aatacctccg
     3301 gaatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     3361 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     3421 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     3481 tgcaggacga ggcagcgcgg ctatcgtggc tggccgcgac gggcgttcct tgcgcagctg
     3541 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     3601 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     3661 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3721 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3781 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     3841 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3901 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3961 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     4021 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     4081 ttgacgagtt cttctgagtc gacaatcaac ctctggatta caaaatttgt gaaagattga
     4141 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     4201 tgtatcatgc gttaactaaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4261 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4321 aaactcatca atgtatctta tcatgtctgg aattgactca aatgatgtca attagtctat
     4381 cagaagctca tctggtctcc cttccggggg acaagacatc cctgtttaat atttaaacag
     4441 cagtgttccc aaactgggtt cttatatccc ttgctctggt caaccaggtt gcagggtttc
     4501 ctgtcctcac aggaacgaag tccctaaaga aacagtggca gccaggttta gccccggaat
     4561 tgactggatt ccttttttag ggcccattgg tatggctttt tccccgtatc cccccaggtg
     4621 tctgcaggct caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc
     4681 ccgtgcccgg gctgtccccg cacgctgccg gctcggggat gcggggggag cgccggaccg
     4741 gagcggagcc ccgggcggct cgctgctgcc ccctagcggg ggagggacgt aattacatcc
     4801 ctgggggctt tggggggggg ctgtccctga tatctataac aagaaaatat atatataata
     4861 agttatcacg taagtagaac atgaaataac aatataatta tcgtatgagt taaatcttaa
     4921 aagtcacgta aaagataatc atgcgtcatt ttgactcacg cggtcgttat agttcaaaat
     4981 cagtgacact taccgcattg acaagcacgc ctcacgggag ctccaagcgg cgactgagat
     5041 gtcctaaatg cacagcgacg gattcgcgct atttagaaag agagagcaat atttcaagaa
     5101 tgcatgcgtc aattttacgc agactatctt tctagggtta atctagctgc atcaggatca
     5161 tatcgtcggg tcttttttcc ggctcagtca tcgcccaagc tggcgctatc tgggcatcgg
     5221 ggaggaagaa gcccgtgcct tttcccgcga ggttgaagcg gcatggaaag agtttgccga
     5281 ggatgactgc tgctgcattg acgttgagcg aaaacgcacg tttaccatga tgattcggga
     5341 aggtgtggcc atgcacgcct ttaacggtga actgttcgtt caggccacct gggataccag
     5401 ttcgtcgcgg cttttccgga cacagttccg gatggtcagc ccgaagcgca tcagcaaccc
     5461 gaacaatacc ggcgacagcc ggaactgccg tgccggtgtg cagattaatg acagcggtgc
     5521 ggcgctggga tattacgtca gcgaggacgg gtatcctggc tggatgccgc agaaatggac
     5581 atggataccc cgtgagttac ccggcgggcg cgcttggcgt aatcatggtc atagctgttt
     5641 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5701 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5761 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5821 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5881 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5941 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6001 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6061 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6121 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6181 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6241 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6301 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6361 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6421 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6481 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6541 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6601 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6661 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6721 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6781 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6841 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6901 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6961 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     7021 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7081 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7141 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7201 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7261 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7321 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7381 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7441 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7501 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7561 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7621 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7681 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7741 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     7801 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     7861 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     7921 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     7981 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     8041 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     8101 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     8161 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     8221 cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     8281 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     8341 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg
     8401 cctcgttcat tcacgttttt gaacccgtgg aggacgggca gactcgcggt gcaaatgtgt
     8461 tttacagcgt gatggagcag atgaagatgc tcgacacgct gcagaacacg cagctagatt
     8521 aaccctagaa agataatcat attgtgacgt acgttaaaga taatcatgtg taaaattgac
     8581 gcatgtgttt tatcggtctg tatatcgagg tttatttatt aatttgaata gatattaagt
     8641 tttattatat ttacacttac atactaataa taaattcaac aaacaattta tttatgttta
     8701 tttatttatt aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca aa
//
