LOCUS       Exported                9080 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Plasmid with 4xMx PylT cassette and MX1201 PylRS for amber 
            suppression; for transient or stable piggyBac-mediated integration.
ACCESSION   .
VERSION     .
KEYWORDS    pAS_4xMx PylT_FLAG-Mx PylRS YA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9080)
  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 9080)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9080
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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        2740..2951
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2964..3232
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             3280..3303
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    4153..4725
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4320..4337)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4547..4566
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4734..5528
                     /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(4788..4807)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5398..5417
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5588..5608)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5650..5771
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5687..5706)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5741..5760
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6534..6568
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7059..7075)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7059..7075)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7072..7094)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7083..7099
                     /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(7107..7137)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7152..7173
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7290..7307)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7461..8049)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7541..7560)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(8220..9080)
                     /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     8843..8862
                     /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 agtggccttg gagctgcagt
     1381 atttagcatg ccccacccat ctgcaaggca ttctggatag tgtcaaaaca gccggaaatc
     1441 aagtccgttt atctcaaact ttagcatttt gggaataaat gatatttgct atgctggtta
     1501 aattagattt tagttaaatt tcctgctgaa gctctagtac gataagtaac ttgacctaag
     1561 tgtaaagttg agatttcctt caggtttata tagcttgtgc gccgcctggg tacctcgggg
     1621 gacggtccgg cgaccagcgg gtctctaaaa cctagccagc ggggttcgac gccccggtct
     1681 ctcggacaag tgcggttttt atcgctgcag tatttagcat gccccaccca tctgcaaggc
     1741 attctggata gtgtcaaaac agccggaaat caagtccgtt tatctcaaac tttagcattt
     1801 tgggaataaa tgatatttgc tatgctggtt aaattagatt ttagttaaat ttcctgctga
     1861 agctctagta cgataagtaa cttgacctaa gtgtaaagtt gagatttcct tcaggtttat
     1921 atagcttgtg cgccgcctgg gtacctcggg ggacggtccg gcgaccagcg ggtctctaaa
     1981 acctagccag cggggttcga cgccccggtc tctcggacaa gtgcggtttt tcttgctgca
     2041 gtatttagca tgccccaccc atctgcaagg cattctggat agtgtcaaaa cagccggaaa
     2101 tcaagtccgt ttatctcaaa ctttagcatt ttgggaataa atgatatttg ctatgctggt
     2161 taaattagat tttagttaaa tttcctgctg aagctctagt acgataagta acttgaccta
     2221 agtgtaaagt tgagatttcc ttcaggttta tatagcttgt gcgccgcctg ggtacctcgg
     2281 gggacggtcc ggcgaccagc gggtctctaa aacctagcca gcggggttcg acgccccggt
     2341 ctctcggaca agtgcggttt ttttgcctgc agtatttagc atgccccacc catctgcaag
     2401 gcattctgga tagtgtcaaa acagccggaa atcaagtccg tttatctcaa actttagcat
     2461 tttgggaata aatgatattt gctatgctgg ttaaattaga ttttagttaa atttcctgct
     2521 gaagctctag tacgataagt aacttgacct aagtgtaaag ttgagatttc cttcaggttt
     2581 atatagcttg tgcgccgcct gggtacctcg ggggacggtc cggcgaccag cgggtctcta
     2641 aaacctagcc agcggggttc gacgccccgg tctctcggac aagtgcggtt tttactagtg
     2701 ctctagaaag gatctgcgat cgctccggtg cccgtcagtg ggcagagcgc acatcgccca
     2761 cagtccccga gaagttgggg ggaggggtcg gcaattgaac gggtgcctag agaaggtggc
     2821 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     2881 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     2941 ccagaacaca gctgaagctt cgaggggctc gcatctctcc ttcacgcgcc cgccgcccta
     3001 cctgaggccg ccatccacgc cggttgagtc gcgttctgcc gcctcccgcc tgtggtgcct
     3061 cctgaactgc gtccgccgtc taggtaagtt taaagctcag gtcgagaccg ggcctttgtc
     3121 cggcgctccc ttggagccta cctagactca gccggctctc cacgctttgc ctgaccctgc
     3181 ttgctcaact ctacgtcttt gtttcgtttt ctgttctgcg ccgttacaga tccaagctgt
     3241 gaccggcgcc tactctagag ctagctcgcg accaccatgg actacaagga cgacgacgac
     3301 aagacggtaa agtatacgga tgcacagata cagcgccttc gcgaatacgg caacgggacc
     3361 tatgagcaga aggtcttcga ggacctcgca tcgagggatg ctgccttctc caaggagatg
     3421 tccgtcgcct ctaccgacaa cgagaagaag atcaagggga tgatcgccaa tccgtcccgt
     3481 catggattga cccagctgat gaacgacatc gccgacgcat tggtcgccga gggtttcatc
     3541 gaggtccgta cgcccatatt catatcgaag gatgcgctgg cacgtatgac catcaccgag
     3601 gacaagcccc ttttcaagca ggtcttctgg atcgacgaga aaagggcgct caggcctatg
     3661 ctggcaccta accttgcctc cgtcatgagg gacctgaggg accatacgga cggtccggtg
     3721 aagatcttcg agatgggttc ctgcttcagg aaggagtccc acagcgggat gcatctggag
     3781 gagttcacca tgctgaacct cgtggacatg ggtccccgcg gagacgccac ggaggtcctg
     3841 aagaactaca tatcggtcgt gatgaaggcg gccggtctcc cggacttcga cctcgtacag
     3901 gaggagtccg acgtatacaa ggagaccata gacgtggaga tcaacggtca ggaggtctgt
     3961 tccgcagccg tcggtccaca ctatctcgat gcggcccacg atgtccacga gccttggtcc
     4021 ggagcgggat tcggtctcga acgcctgctg accatcaggg agaagtacag caccgtgaag
     4081 aagggaggag ccagcatcag ctacctcaac ggtgcgaaga tcaactgatg aatcgatgga
     4141 tccgcggccg cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga
     4201 ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa
     4261 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc
     4321 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc
     4381 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg
     4441 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
     4501 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
     4561 cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct
     4621 ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc
     4681 cccgaaccac ggggacgtgg ttttcctttg aaaaacacga taatacctcc ggaatgattg
     4741 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     4801 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     4861 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     4921 aggcagcgcg gctatcgtgg ctggccgcga cgggcgttcc ttgcgcagct gtgctcgacg
     4981 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     5041 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     5101 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     5161 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     5221 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     5281 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     5341 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     5401 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     5461 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     5521 tcttctgagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     5581 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     5641 cgttaactaa acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5701 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5761 aatgtatctt atcatgtctg gaattgactc aaatgatgtc aattagtcta tcagaagctc
     5821 atctggtctc ccttccgggg gacaagacat ccctgtttaa tatttaaaca gcagtgttcc
     5881 caaactgggt tcttatatcc cttgctctgg tcaaccaggt tgcagggttt cctgtcctca
     5941 caggaacgaa gtccctaaag aaacagtggc agccaggttt agccccggaa ttgactggat
     6001 tcctttttta gggcccattg gtatggcttt ttccccgtat ccccccaggt gtctgcaggc
     6061 tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg
     6121 ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc
     6181 cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct
     6241 ttgggggggg gctgtccctg atatctataa caagaaaata tatatataat aagttatcac
     6301 gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt
     6361 aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac
     6421 ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga tgtcctaaat
     6481 gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt
     6541 caattttacg cagactatct ttctagggtt aatctagctg catcaggatc atatcgtcgg
     6601 gtcttttttc cggctcagtc atcgcccaag ctggcgctat ctgggcatcg gggaggaaga
     6661 agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa gagtttgccg aggatgactg
     6721 ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg atgattcggg aaggtgtggc
     6781 catgcacgcc tttaacggtg aactgttcgt tcaggccacc tgggatacca gttcgtcgcg
     6841 gcttttccgg acacagttcc ggatggtcag cccgaagcgc atcagcaacc cgaacaatac
     6901 cggcgacagc cggaactgcc gtgccggtgt gcagattaat gacagcggtg cggcgctggg
     6961 atattacgtc agcgaggacg ggtatcctgg ctggatgccg cagaaatgga catggatacc
     7021 ccgtgagtta cccggcgggc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     7081 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     7141 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     7201 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     7261 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     7321 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     7381 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     7441 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     7501 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     7561 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     7621 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     7681 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     7741 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     7801 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     7861 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     7921 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     7981 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     8041 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     8101 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     8161 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     8221 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     8281 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     8341 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     8401 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     8461 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     8521 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     8581 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     8641 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     8701 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     8761 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     8821 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     8881 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     8941 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     9001 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     9061 cggaaatgtt gaatactcat
//
