LOCUS       sequence-310400-        9126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid with 4xhybrid PylT cassette (mutant A41AA C55A) and rat 
            CRFR1 95TAG 263TAA, for transient transfection or stable, 
            piggybac-mediated, integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9126)
  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 9126)
  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 CRFR1 95TAG 263TAA-HA
FEATURES             Location/Qualifiers
     source          1..9126
                     /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      2688..2697
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3939..3965
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(3985..4559,4560..4562,4563..4571)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4152..4169)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4379..4398
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4584..4982
                     /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(5048..5068)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5110..5231
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5147..5166)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5201..5220
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5994..6028
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6519..6535)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6519..6535)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6532..6554)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6543..6559
                     /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(6567..6573,6574..6591,6592..6597))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6612..6633
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6750..6767)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6921..7509)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7001..7020)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7680..8471,8472..8540))
                     /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     8303..8322
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8541..8645)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8671..9126)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8808..8829)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     9020..9039
                     /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 agagctagcc accatgggac
     2701 ggcgcccgca gctccggctc gtgaaggccc ttctccttct ggggctgaac cctgtgtcca
     2761 cctcccttca ggatcagcgc tgtgagaacc tgtccctgac cagcaatgtt tctggcctgc
     2821 agtgcaatgc atccgtggac ctcattggca cctgctggcc ccggagccct gcgggccagt
     2881 tggtggtccg accctgccct gcctttttct acggtgtccg ctacaacacg acaaacaatg
     2941 gctaccggga gtgcctggcc aacggcagct gggcatagcg tgtgaattat tctgagtgcc
     3001 aggagattct caacgaagag aagaagagca aagtacacta ccatgttgca gtcatcatca
     3061 actacctggg tcactgcatc tccctggtag ccctcctggt ggcctttgtc ctcttcttgc
     3121 ggctcaggag catccggtgc ctgagaaaca tcatccactg gaacctcatc tcggctttca
     3181 tcctacgcaa cgccacgtgg tttgtggtcc agctcaccgt gagccccgag gtgcaccaga
     3241 gcaatgtggc ctggtgtagg ttggtgacag ccgcctacaa ttacttccat gtaaccaact
     3301 tcttctggat gttcggtgag ggctgctacc tgcacacagc cattgtgctc acgtactcca
     3361 ccgaccgtct gcgcaagtgg atgttcgtct gcattggctg gggtgtacct ttccccatca
     3421 ttgtggcttg ggccattggg aagctgcact acgacaatga aaagtgctgg tttggcaaat
     3481 aacctggggt atacactgac tacatctacc agggccccat gatcctggtc ctgctgatca
     3541 actttatctt tctcttcaac attgtccgca tcctcatgac caaactccgg gcatccacta
     3601 catctgagac cattcagtac aggaaggctg tgaaggccac tctggtgctc ctgccccttc
     3661 tgggcatcac ctacatgttg ttcttcgtca accctggaga ggacgaggtc tccagggtcg
     3721 tcttcatcta cttcaactct tttctggagt cctttcaggg cttctttgtg tctgtgttct
     3781 actgttttct gaacagtgag gtccgctccg ctatccggaa gaggtggcgt cggtggcagg
     3841 acaagcactc catcagagcc cgagtggccc gagctatgtc catccccacc tccccgacca
     3901 gagtcagctt tcacagcatc aagcagtcca cagcagtgta cccatacgat gttccagatt
     3961 acgcatgaag gatccgcggc cgcgcccctc tccctccccc ccccctaacg ttactggccg
     4021 aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc
     4081 gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag
     4141 gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt
     4201 tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa
     4261 ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc
     4321 aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg
     4381 gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat
     4441 gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa
     4501 acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataata
     4561 tggccacaac catggcgtcc ggaatggcca agcctttgtc tcaagaagaa tccaccctca
     4621 ttgaaagagc aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca
     4681 gcgcagctct ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg
     4741 ggggaccttg tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc
     4801 tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt
     4861 gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg
     4921 gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct
     4981 gaccatgggt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     5041 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     5101 cgttaactaa acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5161 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5221 aatgtatctt atcatgtctg gaattgactc aaatgatgtc aattagtcta tcagaagctc
     5281 atctggtctc ccttccgggg gacaagacat ccctgtttaa tatttaaaca gcagtgttcc
     5341 caaactgggt tcttatatcc cttgctctgg tcaaccaggt tgcagggttt cctgtcctca
     5401 caggaacgaa gtccctaaag aaacagtggc agccaggttt agccccggaa ttgactggat
     5461 tcctttttta gggcccattg gtatggcttt ttccccgtat ccccccaggt gtctgcaggc
     5521 tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg
     5581 ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc
     5641 cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct
     5701 ttgggggggg gctgtccctg atatctataa caagaaaata tatatataat aagttatcac
     5761 gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt
     5821 aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac
     5881 ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga tgtcctaaat
     5941 gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt
     6001 caattttacg cagactatct ttctagggtt aatctagctg catcaggatc atatcgtcgg
     6061 gtcttttttc cggctcagtc atcgcccaag ctggcgctat ctgggcatcg gggaggaaga
     6121 agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa gagtttgccg aggatgactg
     6181 ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg atgattcggg aaggtgtggc
     6241 catgcacgcc tttaacggtg aactgttcgt tcaggccacc tgggatacca gttcgtcgcg
     6301 gcttttccgg acacagttcc ggatggtcag cccgaagcgc atcagcaacc cgaacaatac
     6361 cggcgacagc cggaactgcc gtgccggtgt gcagattaat gacagcggtg cggcgctggg
     6421 atattacgtc agcgaggacg ggtatcctgg ctggatgccg cagaaatgga catggatacc
     6481 ccgtgagtta cccggcgggc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     6541 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     6601 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     6661 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     6721 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     6781 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     6841 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6901 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6961 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     7021 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     7081 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     7141 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     7201 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     7261 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     7321 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     7381 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     7441 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     7501 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     7561 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     7621 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     7681 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     7741 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     7801 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     7861 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     7921 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7981 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     8041 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     8101 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     8161 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     8221 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     8281 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     8341 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     8401 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     8461 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     8521 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     8581 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     8641 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
     8701 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     8761 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     8821 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     8881 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     8941 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     9001 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     9061 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     9121 gcgcgt
//
