LOCUS       sequence-311597-        7731 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid with G1 PylRS for amber suppression in transient or stable, 
            piggybac-mediated, integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7731)
  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 7731)
  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_FLAG-G1 PylRS
FEATURES             Location/Qualifiers
     source          1..7731
                     /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        818..1029
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1042..1310
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      1342..1351
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1351..1374
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2218..2790
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2385..2402)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2612..2631
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2799..3593
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLAATGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2853..2872)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3463..3482
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3653..3673)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3715..3836
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3752..3771)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3806..3825
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4599..4633
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5124..5140)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5124..5140)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5137..5159)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5148..5164
                     /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(5172..5178,5179..5196,5197..5202))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5217..5238
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5355..5372)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5526..6114)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5606..5625)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6285..7076,7077..7145))
                     /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     6908..6927
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7146..7250)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7276..7731)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7413..7434)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7625..7644
                     /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 aactagtgct
      781 ctagaaagga tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
      841 gtccccgaga agttgggggg aggggtcggc aattgaacgg gtgcctagag aaggtggcgc
      901 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
      961 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     1021 agaacacagc tgaagcttcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc
     1081 tgaggccgcc atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc
     1141 tgaactgcgt ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg
     1201 gcgctccctt ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt
     1261 gctcaactct acgtctttgt ttcgttttct gttctgcgcc gttacagatc caagctgtga
     1321 ccggcgccta ctctagagct agccaccatg gactacaagg acgacgacga caaggtggtc
     1381 aagttcaccg acagccagat ccagcacctg atggaatacg gcgacaacga ttggagcgag
     1441 gccgagttcg aagatgccgc cgctagagac aaagagttca gcagccagtt cagcaagctg
     1501 aagtccgcca acgacaaggg cctgaaggac gtgatcgcca atcctcggaa cgacctgacc
     1561 gacctggaaa acaagatcag agagaagctg gccgccagag gcttcatcga ggtgcacacc
     1621 cctatcttcg tgtccaagtc tgccctggcc aagatgacca tcaccgagga tcaccctctg
     1681 ttcaaacagg tgttctggat cgacgacaag cgggccctga ggcctatgca cgccatgaac
     1741 ctgtacaaag tgatgagaga gctgcgggac cacacaaagg gccccgtgaa gatctttgag
     1801 atcggcagct gcttccggaa agagagcaag agcagcaccc acctggaaga gttcaccatg
     1861 ctgaacctgg tggaaatggg ccccgacggc gatcctatgg aacacctgaa gatgtatatc
     1921 ggcgacatca tggacgccgt gggcgtcgag tacaccacca gcagagaaga gtccgacgtg
     1981 tacgtggaaa ccctggacgt ggaaatcaac ggcaccgaag tggctagcgg agccgtggga
     2041 cctcacaaac tggatcctgc tcacgacgtg cacgaacctt gggccggaat cggctttggc
     2101 ctggaaagac tgctgatgct gaagaacggc aagagcaacg ccagaaagac cggcaagtcc
     2161 atcacctacc tgaacggcta caagctggac tgatgaatcg atggatccgc ggccgcgccc
     2221 ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc
     2281 gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa
     2341 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat
     2401 gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac
     2461 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg
     2521 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt
     2581 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg
     2641 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac
     2701 atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga
     2761 cgtggttttc ctttgaaaaa cacgataata cctccggaat gattgaacaa gatggattgc
     2821 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     2881 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     2941 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     3001 cgtggctggc cgcgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3061 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3121 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3181 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     3241 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     3301 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     3361 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3421 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     3481 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3541 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagtcgaca
     3601 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     3661 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgcgtta actaaacttg
     3721 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3781 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     3841 gtctggaatt gactcaaatg atgtcaatta gtctatcaga agctcatctg gtctcccttc
     3901 cgggggacaa gacatccctg tttaatattt aaacagcagt gttcccaaac tgggttctta
     3961 tatcccttgc tctggtcaac caggttgcag ggtttcctgt cctcacagga acgaagtccc
     4021 taaagaaaca gtggcagcca ggtttagccc cggaattgac tggattcctt ttttagggcc
     4081 cattggtatg gctttttccc cgtatccccc caggtgtctg caggctcaaa gagcagcgag
     4141 aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg
     4201 ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct
     4261 gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt
     4321 ccctgatatc tataacaaga aaatatatat ataataagtt atcacgtaag tagaacatga
     4381 aataacaata taattatcgt atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc
     4441 gtcattttga ctcacgcggt cgttatagtt caaaatcagt gacacttacc gcattgacaa
     4501 gcacgcctca cgggagctcc aagcggcgac tgagatgtcc taaatgcaca gcgacggatt
     4561 cgcgctattt agaaagagag agcaatattt caagaatgca tgcgtcaatt ttacgcagac
     4621 tatctttcta gggttaatct agctgcatca ggatcatatc gtcgggtctt ttttccggct
     4681 cagtcatcgc ccaagctggc gctatctggg catcggggag gaagaagccc gtgccttttc
     4741 ccgcgaggtt gaagcggcat ggaaagagtt tgccgaggat gactgctgct gcattgacgt
     4801 tgagcgaaaa cgcacgttta ccatgatgat tcgggaaggt gtggccatgc acgcctttaa
     4861 cggtgaactg ttcgttcagg ccacctggga taccagttcg tcgcggcttt tccggacaca
     4921 gttccggatg gtcagcccga agcgcatcag caacccgaac aataccggcg acagccggaa
     4981 ctgccgtgcc ggtgtgcaga ttaatgacag cggtgcggcg ctgggatatt acgtcagcga
     5041 ggacgggtat cctggctgga tgccgcagaa atggacatgg ataccccgtg agttacccgg
     5101 cgggcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5161 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5221 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5281 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5341 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5401 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5461 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5521 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5581 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5641 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5701 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5761 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5821 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5881 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5941 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6001 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6061 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6121 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6181 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6241 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6301 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6361 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6421 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6481 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6541 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6601 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6661 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6721 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6781 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6841 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6901 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6961 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7021 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7081 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7141 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7201 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7261 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     7321 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     7381 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     7441 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     7501 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     7561 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     7621 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     7681 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg t
//
