LOCUS       sequence-310518-        8572 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid with 4xhybrid PylT cassette (Mx1201 G1 PylT hybrid 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 8572)
  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 8572)
  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 A41AA C55A sfGFP 150TAG
FEATURES             Location/Qualifiers
     source          1..8572
                     /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        2164..2375
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2388..2656
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      2690..2699
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(3431..4005,4006..4008,4009..4017)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3598..3615)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3825..3844
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4030..4428
                     /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(4494..4514)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4556..4677
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4593..4612)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4647..4666
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5440..5474
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5965..5981)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5965..5981)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5978..6000)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5989..6005
                     /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(6013..6019,6020..6037,6038..6043))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6058..6079
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6196..6213)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6367..6955)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6447..6466)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7126..7917,7918..7986))
                     /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     7749..7768
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7987..8091)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8117..8572)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8254..8275)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8466..8485
                     /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 ggtgtcgaac cccgctttac aggttttaga
      841 gacccgtttg ctcgccggag cgtcccccga 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 ccgagagacc
     1141 ggggtgtcga accccgcttt acaggtttta gagacccgtt tgctcgccgg agcgtccccc
     1201 gaggtaccca ggcggcgcac aagctatata aacctgaagg aaatctcaac tttacactta
     1261 ggtcaagtta cttatcgtac tagagcttca gcaggaaatt taactaaaat ctaatttaac
     1321 cagcatagca aatatcattt attcccaaaa tgctaaagtt tgagataaac ggacttgatt
     1381 tccggctgtt ttgacactat ccagaatgcc ttgcagatgg gtggggcatg ctaaatactg
     1441 cagcaagaaa aaccgcactt gtccgagaga ccggggtgtc gaaccccgct ttacaggttt
     1501 tagagacccg tttgctcgcc ggagcgtccc ccgaggtacc caggcggcgc acaagctata
     1561 taaacctgaa ggaaatctca actttacact taggtcaagt tacttatcgt actagagctt
     1621 cagcaggaaa tttaactaaa atctaattta accagcatag caaatatcat ttattcccaa
     1681 aatgctaaag tttgagataa acggacttga tttccggctg ttttgacact atccagaatg
     1741 ccttgcagat gggtggggca tgctaaatac tgcagcgata aaaaccgcac ttgtccgaga
     1801 gaccggggtg tcgaaccccg ctttacaggt tttagagacc cgtttgctcg ccggagcgtc
     1861 ccccgaggta cccaggcggc gcacaagcta tataaacctg aaggaaatct caactttaca
     1921 cttaggtcaa gttacttatc gtactagagc ttcagcagga aatttaacta aaatctaatt
     1981 taaccagcat agcaaatatc atttattccc aaaatgctaa agtttgagat aaacggactt
     2041 gatttccggc tgttttgaca ctatccagaa tgccttgcag atgggtgggg catgctaaat
     2101 actgcagctc caaggccact agtgctctag aaaggatctg cgatcgctcc ggtgcccgtc
     2161 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
     2221 gaacgggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     2281 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     2341 ttctttttcg caacgggttt gccgccagaa cacagctgaa gcttcgaggg gctcgcatct
     2401 ctccttcacg cgcccgccgc cctacctgag gccgccatcc acgccggttg agtcgcgttc
     2461 tgccgcctcc cgcctgtggt gcctcctgaa ctgcgtccgc cgtctaggta agtttaaagc
     2521 tcaggtcgag accgggcctt tgtccggcgc tcccttggag cctacctaga ctcagccggc
     2581 tctccacgct ttgcctgacc ctgcttgctc aactctacgt ctttgtttcg ttttctgttc
     2641 tgcgccgtta cagatccaag ctgtgaccgg cgcctactct agagctagcg ccaccatggt
     2701 gtccaagggc gaggaactgt tcaccggcgt ggtgcccatc ctggtggaac tggatggcga
     2761 cgtgaacggc cacaagttct ctgtgcgggg agagggcgaa ggcgacgcca caaatggcaa
     2821 gctgaccctg gagttcatct gcaccaccgg caagctgccc gtgccttggc ctaccctcgt
     2881 gaccacactg acctacggcg tgcagtgctt cagcagatac cccgaccata tgaagcggca
     2941 cgacttcttc aagagcgcca tgcccgaggg ctacgtgcag gaacggacca tcagcttcaa
     3001 ggacgacggc acctacaaga ccagagccga agtgaagttc gagggcgaca ccctcgtgaa
     3061 ccggatcgag ctgaagggca tcgatttcaa agaggacggc aacatcctgg gccacaagct
     3121 ggagtacaac ttcaacagcc actaggtgta catcaccgcc gacaagcaga agaacggcat
     3181 caaggccaac ttcaagatcc ggcacaacgt ggaagatggc agcgtgcagc tggccgacca
     3241 ctaccagcag aacaccccca tcggagatgg ccccgtgctg ctgcccgaca accactacct
     3301 gagcacccag agcgtgctga gcaaggaccc caacgagaag cgggaccaca tggtgctgct
     3361 ggaattcgtg accgccgctg gcatcaccca cggcatggac gagctgtaca agggcagctg
     3421 agcggccgcg cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat
     3481 aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg
     3541 tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc
     3601 tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt
     3661 cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg
     3721 acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac
     3781 cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg
     3841 tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg
     3901 ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc
     3961 cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg
     4021 gcgtccggaa tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg
     4081 gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct
     4141 agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca
     4201 gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc
     4261 gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt
     4321 ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca
     4381 gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctgacc atgggtcgac
     4441 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     4501 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgcgtt aactaaactt
     4561 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4621 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4681 tgtctggaat tgactcaaat gatgtcaatt agtctatcag aagctcatct ggtctccctt
     4741 ccgggggaca agacatccct gtttaatatt taaacagcag tgttcccaaa ctgggttctt
     4801 atatcccttg ctctggtcaa ccaggttgca gggtttcctg tcctcacagg aacgaagtcc
     4861 ctaaagaaac agtggcagcc aggtttagcc ccggaattga ctggattcct tttttagggc
     4921 ccattggtat ggctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga
     4981 gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac
     5041 gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc
     5101 tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg
     5161 tccctgatat ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg
     5221 aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg
     5281 cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca
     5341 agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat
     5401 tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga
     5461 ctatctttct agggttaatc tagctgcatc aggatcatat cgtcgggtct tttttccggc
     5521 tcagtcatcg cccaagctgg cgctatctgg gcatcgggga ggaagaagcc cgtgcctttt
     5581 cccgcgaggt tgaagcggca tggaaagagt ttgccgagga tgactgctgc tgcattgacg
     5641 ttgagcgaaa acgcacgttt accatgatga ttcgggaagg tgtggccatg cacgccttta
     5701 acggtgaact gttcgttcag gccacctggg ataccagttc gtcgcggctt ttccggacac
     5761 agttccggat ggtcagcccg aagcgcatca gcaacccgaa caataccggc gacagccgga
     5821 actgccgtgc cggtgtgcag attaatgaca gcggtgcggc gctgggatat tacgtcagcg
     5881 aggacgggta tcctggctgg atgccgcaga aatggacatg gataccccgt gagttacccg
     5941 gcgggcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     6001 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     6061 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6121 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6181 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     6241 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     6301 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6361 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6421 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6481 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6541 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6601 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6661 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6721 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6781 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     6841 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6901 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6961 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     7021 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     7081 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     7141 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     7201 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     7261 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     7321 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     7381 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     7441 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     7501 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     7561 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     7621 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     7681 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     7741 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     7801 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     7861 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     7921 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7981 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     8041 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     8101 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     8161 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     8221 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     8281 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     8341 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     8401 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     8461 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     8521 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gt
//
