LOCUS       sequence-309867-        8579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid with 4xG1 PylT cassette (G1 PylT mutant A41AA C55A) 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 8579)
  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 8579)
  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 A41AA C55A sfGFP 150TAG
FEATURES             Location/Qualifiers
     source          1..8579
                     /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        2171..2382
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2395..2663
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      2697..2706
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(3438..4012,4013..4015,4016..4024)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3605..3622)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3832..3851
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4037..4435
                     /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(4501..4521)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4563..4684
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4600..4619)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4654..4673
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5447..5481
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5972..5988)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5972..5988)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5985..6007)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5996..6012
                     /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(6020..6026,6027..6044,6045..6050))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6065..6086
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6203..6220)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6374..6962)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6454..6473)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7133..7924,7925..7993))
                     /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     7756..7775
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7994..8098)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8124..8579)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8261..8282)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8473..8492
                     /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 acgtaaaaac cgcacttgtc cgaaaggccg gggtgtcgaa ccccgcttta caggttttag
      841 agacccgttt gctcgccgga gcgccctccg aggtacccag gcggcgcaca agctatataa
      901 acctgaagga aatctcaact ttacacttag gtcaagttac ttatcgtact agagcttcag
      961 caggaaattt aactaaaatc taatttaacc agcatagcaa atatcattta ttcccaaaat
     1021 gctaaagttt gagataaacg gacttgattt ccggctgttt tgacactatc cagaatgcct
     1081 tgcagatggg tggggcatgc taaatactgc aggcaaaaaa accgcacttg tccgaaaggc
     1141 cggggtgtcg aaccccgctt tacaggtttt agagacccgt ttgctcgccg gagcgccctc
     1201 cgaggtaccc aggcggcgca caagctatat aaacctgaag gaaatctcaa ctttacactt
     1261 aggtcaagtt acttatcgta ctagagcttc agcaggaaat ttaactaaaa tctaatttaa
     1321 ccagcatagc aaatatcatt tattcccaaa atgctaaagt ttgagataaa cggacttgat
     1381 ttccggctgt tttgacacta tccagaatgc cttgcagatg ggtggggcat gctaaatact
     1441 gcagcaagaa aaaccgcact tgtccgaaag gccggggtgt cgaaccccgc tttacaggtt
     1501 ttagagaccc gtttgctcgc cggagcgccc tccgaggtac ccaggcggcg cacaagctat
     1561 ataaacctga aggaaatctc aactttacac ttaggtcaag ttacttatcg tactagagct
     1621 tcagcaggaa atttaactaa aatctaattt aaccagcata gcaaatatca tttattccca
     1681 aaatgctaaa gtttgagata aacggacttg atttccggct gttttgacac tatccagaat
     1741 gccttgcaga tgggtggggc atgctaaata ctgcagcgat aaaaaccgca cttgtccgaa
     1801 aggccggggt gtcgaacccc gctttacagg ttttagagac ccgtttgctc gccggagcgc
     1861 cctccgaggt acccaggcgg cgcacaagct atataaacct gaaggaaatc tcaactttac
     1921 acttaggtca agttacttat cgtactagag cttcagcagg aaatttaact aaaatctaat
     1981 ttaaccagca tagcaaatat catttattcc caaaatgcta aagtttgaga taaacggact
     2041 tgatttccgg ctgttttgac actatccaga atgccttgca gatgggtggg gcatgctaaa
     2101 tactgcagct ccaagcctag ggccactagt gctctagaaa ggatctgcga tcgctccggt
     2161 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     2221 ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     2281 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     2341 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac agctgaagct tcgaggggct
     2401 cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt
     2461 cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt
     2521 ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc
     2581 agccggctct ccacgctttg cctgaccctg cttgctcaac tctacgtctt tgtttcgttt
     2641 tctgttctgc gccgttacag atccaagctg tgaccggcgc ctactctaga gctagcgcca
     2701 ccatggtgtc caagggcgag gaactgttca ccggcgtggt gcccatcctg gtggaactgg
     2761 atggcgacgt gaacggccac aagttctctg tgcggggaga gggcgaaggc gacgccacaa
     2821 atggcaagct gaccctggag ttcatctgca ccaccggcaa gctgcccgtg ccttggccta
     2881 ccctcgtgac cacactgacc tacggcgtgc agtgcttcag cagatacccc gaccatatga
     2941 agcggcacga cttcttcaag agcgccatgc ccgagggcta cgtgcaggaa cggaccatca
     3001 gcttcaagga cgacggcacc tacaagacca gagccgaagt gaagttcgag ggcgacaccc
     3061 tcgtgaaccg gatcgagctg aagggcatcg atttcaaaga ggacggcaac atcctgggcc
     3121 acaagctgga gtacaacttc aacagccact aggtgtacat caccgccgac aagcagaaga
     3181 acggcatcaa ggccaacttc aagatccggc acaacgtgga agatggcagc gtgcagctgg
     3241 ccgaccacta ccagcagaac acccccatcg gagatggccc cgtgctgctg cccgacaacc
     3301 actacctgag cacccagagc gtgctgagca aggaccccaa cgagaagcgg gaccacatgg
     3361 tgctgctgga attcgtgacc gccgctggca tcacccacgg catggacgag ctgtacaagg
     3421 gcagctgagc ggccgcgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc
     3481 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt
     3541 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt
     3601 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg
     3661 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca
     3721 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg
     3781 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc
     3841 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct
     3901 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta
     3961 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac
     4021 aaccatggcg tccggaatgg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag
     4081 agcaacggct acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc
     4141 tctctctagc gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc
     4201 ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg
     4261 tatcgtcgcg atcggaaatg agaacagggg catcttgagc ccctgcggac ggtgccgaca
     4321 ggtgcttctc gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc
     4381 gacggcagtt gggattcgtg aattgctgcc ctctggttat gtgtgggagg gctgaccatg
     4441 ggtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     4501 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgcgttaac
     4561 taaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4621 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4681 cttatcatgt ctggaattga ctcaaatgat gtcaattagt ctatcagaag ctcatctggt
     4741 ctcccttccg ggggacaaga catccctgtt taatatttaa acagcagtgt tcccaaactg
     4801 ggttcttata tcccttgctc tggtcaacca ggttgcaggg tttcctgtcc tcacaggaac
     4861 gaagtcccta aagaaacagt ggcagccagg tttagccccg gaattgactg gattcctttt
     4921 ttagggccca ttggtatggc tttttccccg tatcccccca ggtgtctgca ggctcaaaga
     4981 gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc
     5041 cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc
     5101 ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg
     5161 ggggctgtcc ctgatatcta taacaagaaa atatatatat aataagttat cacgtaagta
     5221 gaacatgaaa taacaatata attatcgtat gagttaaatc ttaaaagtca cgtaaaagat
     5281 aatcatgcgt cattttgact cacgcggtcg ttatagttca aaatcagtga cacttaccgc
     5341 attgacaagc acgcctcacg ggagctccaa gcggcgactg agatgtccta aatgcacagc
     5401 gacggattcg cgctatttag aaagagagag caatatttca agaatgcatg cgtcaatttt
     5461 acgcagacta tctttctagg gttaatctag ctgcatcagg atcatatcgt cgggtctttt
     5521 ttccggctca gtcatcgccc aagctggcgc tatctgggca tcggggagga agaagcccgt
     5581 gccttttccc gcgaggttga agcggcatgg aaagagtttg ccgaggatga ctgctgctgc
     5641 attgacgttg agcgaaaacg cacgtttacc atgatgattc gggaaggtgt ggccatgcac
     5701 gcctttaacg gtgaactgtt cgttcaggcc acctgggata ccagttcgtc gcggcttttc
     5761 cggacacagt tccggatggt cagcccgaag cgcatcagca acccgaacaa taccggcgac
     5821 agccggaact gccgtgccgg tgtgcagatt aatgacagcg gtgcggcgct gggatattac
     5881 gtcagcgagg acgggtatcc tggctggatg ccgcagaaat ggacatggat accccgtgag
     5941 ttacccggcg ggcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     6001 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     6061 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     6121 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     6181 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     6241 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     6301 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     6361 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     6421 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     6481 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     6541 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     6601 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6661 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6721 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6781 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6841 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6901 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6961 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     7021 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     7081 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     7141 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     7201 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     7261 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     7321 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     7381 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     7441 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     7501 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     7561 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7621 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7681 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7741 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7801 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7861 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7921 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7981 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     8041 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     8101 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     8161 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     8221 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     8281 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     8341 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     8401 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     8461 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     8521 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgt
//
