LOCUS       sequence-305123-        8568 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid with 4xhybrid PylT cassette (Mx1201 G1 PylT hybrid ) 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 8568)
  AUTHORS   Meineke B, Heimgartner J, Eirich J, Landreh M, Elsasser SJ
  TITLE     Site-Specific Incorporation of Two ncAAs for Two-Color Bioorthogonal
            Labeling and Crosslinking of Proteins on Live Mammalian Cells.
  JOURNAL   Cell Rep. 2020 Jun 23;31(12):107811. doi: 
            10.1016/j.celrep.2020.107811.
  PUBMED    32579937
REFERENCE   2  (bases 1 to 8568)
  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: "Cell Rep.";
            date: "2020-06-23"; volume: "31(12):107811. doi"; pages: " 
            10.1016/j.celrep.2020.107811"
COMMENT     Sequence Label: pAS_4xhyb PylT sfGFP 150TAG
FEATURES             Location/Qualifiers
     source          1..8568
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     127..149
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     141..158
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     142..158
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   288..350
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2160..2371
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2384..2652
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      2686..2695
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(3427..4001,4002..4004,4005..4013)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3594..3611)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3821..3840
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4026..4424
                     /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(4490..4510)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4552..4673
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4589..4608)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4643..4662
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5436..5470
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5961..5977)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5961..5977)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5974..5996)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5985..6001
                     /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(6009..6015,6016..6033,6034..6039))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6054..6075
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6192..6209)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6363..6951)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6443..6462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7122..7913,7914..7982))
                     /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     7745..7764
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7983..8087)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8113..8568)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8250..8271)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8462..8481
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
       61 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      121 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcctcg ttcattcacg
      181 tttttgaacc cgtggaggac gggcagactc gcggtgcaaa tgtgttttac agcgtgatgg
      241 agcagatgaa gatgctcgac acgctgcaga acacgcagct agattaaccc tagaaagata
      301 atcatattgt gacgtacgtt aaagataatc atgtgtaaaa ttgacgcatg tgttttatcg
      361 gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat tatatttaca
      421 cttacatact aataataaat tcaacaaaca atttatttat gtttatttat ttattaaaaa
      481 aaacaaaaac tcaaaatttc ttctataaag taacaaaact tttatgaggg acagcccccc
      541 cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc agcgagccgc
      601 ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga
      661 cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt ctcgctgctc
      721 tttgagcctg cagacacctg gggggatacg gggaaaaggc ctccacggcc aactagtgag
      781 acgaaaaacc gcacttgtcc gagagaccgg ggggtcgaac cccgcttaca ggttttagag
      841 acccgtttgc tcgccggagc gtcccccgag gtacccaggc ggcgcacaag ctatataaac
      901 ctgaaggaaa tctcaacttt acacttaggt caagttactt atcgtactag agcttcagca
      961 ggaaatttaa ctaaaatcta atttaaccag catagcaaat atcatttatt cccaaaatgc
     1021 taaagtttga gataaacgga cttgatttcc ggctgttttg acactatcca gaatgccttg
     1081 cagatgggtg gggcatgcta aatactgcag gcaaaaaaac cgcacttgtc cgagagaccg
     1141 gggggtcgaa ccccgcttac aggttttaga gacccgtttg ctcgccggag cgtcccccga
     1201 ggtacccagg cggcgcacaa gctatataaa cctgaaggaa atctcaactt tacacttagg
     1261 tcaagttact tatcgtacta gagcttcagc aggaaattta actaaaatct aatttaacca
     1321 gcatagcaaa tatcatttat tcccaaaatg ctaaagtttg agataaacgg acttgatttc
     1381 cggctgtttt gacactatcc agaatgcctt gcagatgggt ggggcatgct aaatactgca
     1441 gcaagaaaaa ccgcacttgt ccgagagacc ggggggtcga accccgctta caggttttag
     1501 agacccgttt gctcgccgga gcgtcccccg aggtacccag gcggcgcaca agctatataa
     1561 acctgaagga aatctcaact ttacacttag gtcaagttac ttatcgtact agagcttcag
     1621 caggaaattt aactaaaatc taatttaacc agcatagcaa atatcattta ttcccaaaat
     1681 gctaaagttt gagataaacg gacttgattt ccggctgttt tgacactatc cagaatgcct
     1741 tgcagatggg tggggcatgc taaatactgc agcgataaaa accgcacttg tccgagagac
     1801 cggggggtcg aaccccgctt acaggtttta gagacccgtt tgctcgccgg agcgtccccc
     1861 gaggtaccca ggcggcgcac aagctatata aacctgaagg aaatctcaac tttacactta
     1921 ggtcaagtta cttatcgtac tagagcttca gcaggaaatt taactaaaat ctaatttaac
     1981 cagcatagca aatatcattt attcccaaaa tgctaaagtt tgagataaac ggacttgatt
     2041 tccggctgtt ttgacactat ccagaatgcc ttgcagatgg gtggggcatg ctaaatactg
     2101 cagctccaag gccactagtg ctctagaaag gatctgcgat cgctccggtg cccgtcagtg
     2161 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
     2221 gggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
     2281 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
     2341 ttttcgcaac gggtttgccg ccagaacaca gctgaagctt cgaggggctc gcatctctcc
     2401 ttcacgcgcc cgccgcccta cctgaggccg ccatccacgc cggttgagtc gcgttctgcc
     2461 gcctcccgcc tgtggtgcct cctgaactgc gtccgccgtc taggtaagtt taaagctcag
     2521 gtcgagaccg ggcctttgtc cggcgctccc ttggagccta cctagactca gccggctctc
     2581 cacgctttgc ctgaccctgc ttgctcaact ctacgtcttt gtttcgtttt ctgttctgcg
     2641 ccgttacaga tccaagctgt gaccggcgcc tactctagag ctagcgccac catggtgtcc
     2701 aagggcgagg aactgttcac cggcgtggtg cccatcctgg tggaactgga tggcgacgtg
     2761 aacggccaca agttctctgt gcggggagag ggcgaaggcg acgccacaaa tggcaagctg
     2821 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cttggcctac cctcgtgacc
     2881 acactgacct acggcgtgca gtgcttcagc agataccccg accatatgaa gcggcacgac
     2941 ttcttcaaga gcgccatgcc cgagggctac gtgcaggaac ggaccatcag cttcaaggac
     3001 gacggcacct acaagaccag agccgaagtg aagttcgagg gcgacaccct cgtgaaccgg
     3061 atcgagctga agggcatcga tttcaaagag gacggcaaca tcctgggcca caagctggag
     3121 tacaacttca acagccacta ggtgtacatc accgccgaca agcagaagaa cggcatcaag
     3181 gccaacttca agatccggca caacgtggaa gatggcagcg tgcagctggc cgaccactac
     3241 cagcagaaca cccccatcgg agatggcccc gtgctgctgc ccgacaacca ctacctgagc
     3301 acccagagcg tgctgagcaa ggaccccaac gagaagcggg accacatggt gctgctggaa
     3361 ttcgtgaccg ccgctggcat cacccacggc atggacgagc tgtacaaggg cagctgagcg
     3421 gccgcgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg
     3481 ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag
     3541 ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc
     3601 caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg
     3661 aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag
     3721 gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca
     3781 gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt
     3841 caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc
     3901 tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa
     3961 ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca accatggcgt
     4021 ccggaatggc caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta
     4081 caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg
     4141 acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac
     4201 tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga
     4261 tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg
     4321 atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg
     4381 ggattcgtga attgctgccc tctggttatg tgtgggaggg ctgaccatgg gtcgacaatc
     4441 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     4501 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgcgttaact aaacttgttt
     4561 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4621 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4681 tggaattgac tcaaatgatg tcaattagtc tatcagaagc tcatctggtc tcccttccgg
     4741 gggacaagac atccctgttt aatatttaaa cagcagtgtt cccaaactgg gttcttatat
     4801 cccttgctct ggtcaaccag gttgcagggt ttcctgtcct cacaggaacg aagtccctaa
     4861 agaaacagtg gcagccaggt ttagccccgg aattgactgg attccttttt tagggcccat
     4921 tggtatggct ttttccccgt atccccccag gtgtctgcag gctcaaagag cagcgagaag
     4981 cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg
     5041 ccggctcggg gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct
     5101 gccccctagc gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc
     5161 tgatatctat aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat
     5221 aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc
     5281 attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca
     5341 cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc
     5401 gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat
     5461 ctttctaggg ttaatctagc tgcatcagga tcatatcgtc gggtcttttt tccggctcag
     5521 tcatcgccca agctggcgct atctgggcat cggggaggaa gaagcccgtg ccttttcccg
     5581 cgaggttgaa gcggcatgga aagagtttgc cgaggatgac tgctgctgca ttgacgttga
     5641 gcgaaaacgc acgtttacca tgatgattcg ggaaggtgtg gccatgcacg cctttaacgg
     5701 tgaactgttc gttcaggcca cctgggatac cagttcgtcg cggcttttcc ggacacagtt
     5761 ccggatggtc agcccgaagc gcatcagcaa cccgaacaat accggcgaca gccggaactg
     5821 ccgtgccggt gtgcagatta atgacagcgg tgcggcgctg ggatattacg tcagcgagga
     5881 cgggtatcct ggctggatgc cgcagaaatg gacatggata ccccgtgagt tacccggcgg
     5941 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     6001 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     6061 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     6121 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     6181 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     6241 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     6301 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6361 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6421 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6481 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6541 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6601 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6661 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6721 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6781 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     6841 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6901 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6961 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     7021 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     7081 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     7141 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     7201 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     7261 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7321 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7381 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7441 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7501 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7561 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     7621 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7681 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     7741 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7801 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7861 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7921 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7981 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     8041 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     8101 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     8161 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     8221 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     8281 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     8341 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     8401 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     8461 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     8521 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgt
//
