LOCUS       sequence-309635-        8569 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid with 4xG1 PylT cassette 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 8569)
  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 8569)
  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_4xG1 PylT sfGFP 150TAG
FEATURES             Location/Qualifiers
     source          1..8569
                     /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        2161..2372
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2385..2653
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      2687..2696
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(3428..4002,4003..4005,4006..4014)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3595..3612)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3822..3841
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4027..4425
                     /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(4491..4511)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4553..4674
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4590..4609)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4644..4663
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5437..5471
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5962..5978)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5962..5978)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5975..5997)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5986..6002
                     /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(6010..6016,6017..6034,6035..6040))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6055..6076
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6193..6210)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6364..6952)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6444..6463)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7123..7914,7915..7983))
                     /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     7746..7765
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7984..8088)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8114..8569)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8251..8272)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8463..8482
                     /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 aactagtgng
      781 acgaaaaacc gcacttgtcc garaggccgg ggggtcgaac cccgcttnac aggttttaga
      841 gacccgtttg ctcgccggag cgccctccga ggtacccagg cggcgcacaa gctatataaa
      901 cctgaaggaa atctcaactt tacacttagg tcaagttact tatcgtacta gagcttcagc
      961 aggaaattta actaaaatct aatttaacca gcatagcaaa tatcatttat tcccaaaatg
     1021 ctaaagtttg agataaacgg acttgatttc cggctgtttt gacactatcc agaatgcctt
     1081 gcagatgggt ggggcatgct aaatactgca ggcaaaaaaa ccgcacttgt ccgaaaggcc
     1141 ggggggtcga accccgctta caggttttag agacccgttt gctcgccgga gcgccctccg
     1201 aggtacccag gcggcgcaca agctatataa acctgaagga aatctcaact ttacacttag
     1261 gtcaagttac ttatcgtact agagcttcag caggaaattt aactaaaatc taatttaacc
     1321 agcatagcaa atatcattta ttcccaaaat gctaaagttt gagataaacg gacttgattt
     1381 ccggctgttt tgacactatc cagaatgcct tgcagatggg tggggcatgc taaatactgc
     1441 agcaagaaaa accgcacttg tccgaaaggc cggggggtcg aaccccgctt acaggtttta
     1501 gagacccgtt tgctcgccgg agcgccctcc gaggtaccca ggcggcgcac aagctatata
     1561 aacctgaagg aaatctcaac tttacactta ggtcaagtta cttatcgtac tagagcttca
     1621 gcaggaaatt taactaaaat ctaatttaac cagcatagca aatatcattt attcccaaaa
     1681 tgctaaagtt tgtgataaac ggacttgatt tccggctgtt ttgacactat ccagaatgcc
     1741 ttgcagatgg gtggggcatg ctaaatactg cagcgataaa aaccgcactt gtccgaaagg
     1801 ccggggggtc gaaccccgct tacaggtttt agagacccgt ttgctcgccg gagcgccctc
     1861 cgaggtaccc aggcggcgca caagctatat aaacctgaag gaaatctcaa ctttacactt
     1921 aggtcaagtt acttatcgta ctagagcttc agcaggaaat ttaactaaaa tctaatttaa
     1981 ccagcatagc aaatatcatt tattcccaaa atgctaaagt ttgagataaa cggacttgat
     2041 ttccggctgt tttgacacta tccagaatgc cttgcagatg ggtggggcat gctaaatact
     2101 gcagctccaa ggccactagt gctctagaaa ggatctgcga tcgctccggt gcccgtcagt
     2161 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
     2221 cgggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     2281 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     2341 tttttcgcaa cgggtttgcc gccagaacac agctgaagct tcgaggggct cgcatctctc
     2401 cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt cgcgttctgc
     2461 cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt ttaaagctca
     2521 ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc agccggctct
     2581 ccacgctttg cctgaccctg cttgctcaac tctacgtctt tgtttcgttt tctgttctgc
     2641 gccgttacag atccaagctg tgaccggcgc ctactctaga gctagcgcca ccatggtgtc
     2701 caagggcgag gaactgttca ccggcgtggt gcccatcctg gtggaactgg atggcgacgt
     2761 gaacggccac aagttctctg tgcggggaga gggcgaaggc gacgccacaa atggcaagct
     2821 gaccctggag ttcatctgca ccaccggcaa gctgcccgtg ccttggccta ccctcgtgac
     2881 cacactgacc tacggcgtgc agtgcttcag cagatacccc gaccatatga agcggcacga
     2941 cttcttcaag agcgccatgc ccgagggcta cgtgcaggaa cggaccatca gcttcaagga
     3001 cgacggcacc tacaagacca gagccgaagt gaagttcgag ggcgacaccc tcgtgaaccg
     3061 gatcgagctg aagggcatcg atttcaaaga ggacggcaac atcctgggcc acaagctgga
     3121 gtacaacttc aacagccact aggtgtacat caccgccgac aagcagaaga acggcatcaa
     3181 ggccaacttc aagatccggc acaacgtgga agatggcagc gtgcagctgg ccgaccacta
     3241 ccagcagaac acccccatcg gagatggccc cgtgctgctg cccgacaacc actacctgag
     3301 cacccagagc gtgctgagca aggaccccaa cgagaagcgg gaccacatgg tgctgctgga
     3361 attcgtgacc gccgctggca tcacccacgg catggacgag ctgtacaagg gcagctgagc
     3421 ggccgcgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag
     3481 gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga
     3541 gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg
     3601 ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt
     3661 gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca
     3721 ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc
     3781 agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat
     3841 tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc
     3901 ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga
     3961 accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggcg
     4021 tccggaatgg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag agcaacggct
     4081 acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc tctctctagc
     4141 gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa
     4201 ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg tatcgtcgcg
     4261 atcggaaatg agaacagggg catcttgagc ccctgcggac ggtgccgaca ggtgcttctc
     4321 gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc gacggcagtt
     4381 gggattcgtg aattgctgcc ctctggttat gtgtgggagg gctgaccatg ggtcgacaat
     4441 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     4501 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgcgttaac taaacttgtt
     4561 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4621 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4681 ctggaattga ctcaaatgat gtcaattagt ctatcagaag ctcatctggt ctcccttccg
     4741 ggggacaaga catccctgtt taatatttaa acagcagtgt tcccaaactg ggttcttata
     4801 tcccttgctc tggtcaacca ggttgcaggg tttcctgtcc tcacaggaac gaagtcccta
     4861 aagaaacagt ggcagccagg tttagccccg gaattgactg gattcctttt ttagggccca
     4921 ttggtatggc tttttccccg tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa
     4981 gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct
     5041 gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc ggctcgctgc
     5101 tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg ggggctgtcc
     5161 ctgatatcta taacaagaaa atatatatat aataagttat cacgtaagta gaacatgaaa
     5221 taacaatata attatcgtat gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt
     5281 cattttgact cacgcggtcg ttatagttca aaatcagtga cacttaccgc attgacaagc
     5341 acgcctcacg ggagctccaa gcggcgactg agatgtccta aatgcacagc gacggattcg
     5401 cgctatttag aaagagagag caatatttca agaatgcatg cgtcaatttt acgcagacta
     5461 tctttctagg gttaatctag ctgcatcagg atcatatcgt cgggtctttt ttccggctca
     5521 gtcatcgccc aagctggcgc tatctgggca tcggggagga agaagcccgt gccttttccc
     5581 gcgaggttga agcggcatgg aaagagtttg ccgaggatga ctgctgctgc attgacgttg
     5641 agcgaaaacg cacgtttacc atgatgattc gggaaggtgt ggccatgcac gcctttaacg
     5701 gtgaactgtt cgttcaggcc acctgggata ccagttcgtc gcggcttttc cggacacagt
     5761 tccggatggt cagcccgaag cgcatcagca acccgaacaa taccggcgac agccggaact
     5821 gccgtgccgg tgtgcagatt aatgacagcg gtgcggcgct gggatattac gtcagcgagg
     5881 acgggtatcc tggctggatg ccgcagaaat ggacatggat accccgtgag ttacccggcg
     5941 ggcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     6001 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     6061 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6121 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6181 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     6241 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     6301 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     6361 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     6421 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     6481 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     6541 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6601 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6661 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6721 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6781 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     6841 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6901 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6961 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     7021 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     7081 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     7141 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     7201 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     7261 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     7321 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     7381 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     7441 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     7501 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     7561 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     7621 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     7681 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7741 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7801 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7861 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     7921 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     7981 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     8041 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     8101 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     8161 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     8221 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     8281 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     8341 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     8401 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     8461 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     8521 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgt
//
