LOCUS       sequence-344541-        8615 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  amber and ochre dual suppression reporter sfGFP 102TAG 150 TAA 
            expression, with BstTyr(CUA) amber suppressor tRNA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8615)
  AUTHORS   Meineke B, Heimgartner J, Caridha R, Block MF, Kimler KJ, Pires MF, 
            Landreh M, Elsasser SJ
  TITLE     Dual stop codon suppression in mammalian cells with genomically 
            integrated genetic code expansion machinery.
  JOURNAL   Cell Rep Methods. 2023 Nov 20;3(11):100626. doi: 
            10.1016/j.crmeth.2023.100626. Epub 2023 Nov 6.
  PUBMED    37935196
REFERENCE   2  (bases 1 to 8615)
  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 
            Methods."; date: "2023-11-20"; volume: "3(11):100626. doi"; pages: "
            10.1016/j.crmeth.2023.100626. Epub 2023 Nov 6"
COMMENT     Sequence Label: pAS_4xKBstTyrT(CUA)_EF1_sfGFP 102TAG 150TAA
FEATURES             Location/Qualifiers
     source          1..8615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(91..546)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(228..249)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     440..459
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     673..695
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     687..704
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     688..704
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   834..896
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2753..2964
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2977..3245
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      3279..3288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(4020..4594,4595..4597,4598..4606)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4187..4204)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4414..4433
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4619..5017
                     /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(5083..5103)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5145..5266
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5182..5201)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5236..5255
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6029..6063
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6554..6570)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6554..6570)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6567..6589)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6578..6594
                     /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(6602..6608,6609..6626,6627..6632))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6647..6668
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6785..6802)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6956..7544)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7036..7055)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7715..8506,8507..8575))
                     /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     8338..8357
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8576..8615,1..65))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
       61 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
      121 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
      181 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
      241 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
      301 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
      361 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
      421 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
      481 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
      541 gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
      601 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
      661 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcctcgttca
      721 ttcacgtttt tgaacccgtg gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg
      781 tgatggagca gatgaagatg ctcgacacgc tgcagaacac gcagctagat taaccctaga
      841 aagataatca tattgtgacg tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt
      901 ttatcggtct gtatatcgag gtttatttat taatttgaat agatattaag ttttattata
      961 tttacactta catactaata ataaattcaa caaacaattt atttatgttt atttatttat
     1021 taaaaaaaac aaaaactcaa aatttcttct ataaagtaac aaaactttta tgagggacag
     1081 ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg
     1141 agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg
     1201 cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg
     1261 ctgctctttg agcctgcaga cacctggggg gatacgggga aaaggcctcc acggccaact
     1321 agtaaaaacc gcacttgtct ggagggggac ggattcgaac cgccgaaccc aaagggagcg
     1381 gatttagagt ccgccgcgtt tagccacttc gctacccctc cgaggtaccc aggcggcgca
     1441 caagctatat aaacctgaag gaaatctcaa ctttacactt aggtcaagtt acttatcgta
     1501 ctagagcttc agcaggaaat ttaactaaaa tctaatttaa ccagcatagc aaatatcatt
     1561 tattcccaaa atgctaaagt ttgagataaa cggacttgat ttccggctgt tttgacacta
     1621 tccagaatgc cttgcagatg ggtggggcat gctaaatact gcaggaaaaa ccgcacttgt
     1681 ccggaggggg acggattcga accgccgaac ccaaagggag cggatttaga gtccgccgcg
     1741 tttagccact tcgctacccc tccgaggtac ccaggcggcg cacaagctat ataaacctga
     1801 aggaaatctc aactttacac ttaggtcaag ttacttatcg tactagagct tcagcaggaa
     1861 atttaactaa aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa
     1921 gtttgagata aacggacttg atttccggct gttttgacac tatccagaat gccttgcaga
     1981 tgggtggggc atgctaaata ctgcagcatg aaaaaccgca cttgtctgga gggggacgga
     2041 ttcgaaccgc cgaacccaaa gggagcggat ttagagtccg ccgcgtttag ccacttcgct
     2101 acccctccga ggtacccagg cggcgcacaa gctatataaa cctgaaggaa atctcaactt
     2161 tacacttagg tcaagttact tatcgtacta gagcttcagc aggaaattta actaaaatct
     2221 aatttaacca gcatagcaaa tatcatttat tcccaaaatg ctaaagtttg agataaacgg
     2281 acttgatttc cggctgtttt gacactatcc agaatgcctt gcagatgggt ggggcatgct
     2341 aaatactgca gcgataaaaa ccgcacttgt ctggaggggg acggattcga accgccgaac
     2401 ccaaagggag cggatttaga gtccgccgcg tttagccact tcgctacccc tccgaggtac
     2461 ccaggcggcg cacaagctat ataaacctga aggaaatctc aactttacac ttaggtcaag
     2521 ttacttatcg tactagagct tcagcaggaa atttaactaa aatctaattt aaccagcata
     2581 gcaaatatca tttattccca aaatgctaaa gtttgagata aacggacttg atttccggct
     2641 gttttgacac tatccagaat gccttgcaga tgggtggggc atgctaaata ctgcagctcc
     2701 aaggccacta gtgctctaga aaggatctgc gatcgctccg gtgcccgtca gtgggcagag
     2761 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc
     2821 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
     2881 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
     2941 aacgggtttg ccgccagaac acagctgaag cttcgagggg ctcgcatctc tccttcacgc
     3001 gcccgccgcc ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc
     3061 gcctgtggtg cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga
     3121 ccgggccttt gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt
     3181 tgcctgaccc tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac
     3241 agatccaagc tgtgaccggc gcctactcta gagctagcgc caccatggtg tccaagggcg
     3301 aggaactgtt caccggcgtg gtgcccatcc tggtggaact ggatggcgac gtgaacggcc
     3361 acaagttctc tgtgcgggga gagggcgaag gcgacgccac aaatggcaag ctgaccctga
     3421 agttcatctg caccaccggc aagctgcccg tgccttggcc taccctcgtg accacactga
     3481 cctacggcgt gcagtgcttc agcagatacc ccgaccatat gaagcggcac gacttcttca
     3541 agagcgccat gcccgagggc tacgtgcagg aacggaccat cagcttctag gacgacggca
     3601 cctacaagac cagagccgaa gtgaagttcg agggcgacac cctcgtgaac cggatcgagc
     3661 tgaagggcat cgatttcaaa gaggacggca acatcctggg ccacaagctg gagtacaact
     3721 tcaacagcca ctaagtgtac atcaccgccg acaagcagaa gaacggcatc aaggccaact
     3781 tcaagatccg gcacaacgtg gaagatggca gcgtgcagct ggccgaccac taccagcaga
     3841 acacccccat cggagatggc cccgtgctgc tgcccgacaa ccactacctg agcacccaga
     3901 gcgtgctgag caaggacccc aacgagaagc gggaccacat ggtgctgctg gaattcgtga
     3961 ccgccgctgg catcacccac ggcatggacg agctgtacaa gggcagctga gcggccgcgc
     4021 ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt
     4081 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg
     4141 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga
     4201 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa
     4261 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc
     4321 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac
     4381 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag
     4441 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc
     4501 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg
     4561 gacgtggttt tcctttgaaa aacacgatga taatatggcc acaaccatgg cgtccggaat
     4621 ggccaagcct ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa
     4681 cagcatcccc atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg
     4741 catcttcact ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt
     4801 gctgggcact gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa
     4861 tgagaacagg ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca
     4921 tcctgggatc aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg
     4981 tgaattgctg ccctctggtt atgtgtggga gggctgacca tgggtcgaca atcaacctct
     5041 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     5101 atgtggatac gctgctttaa tgcctttgta tcatgcgtta actaaacttg tttattgcag
     5161 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5221 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggaatt
     5281 gactcaaatg atgtcaatta gtctatcaga agctcatctg gtctcccttc cgggggacaa
     5341 gacatccctg tttaatattt aaacagcagt gttcccaaac tgggttctta tatcccttgc
     5401 tctggtcaac caggttgcag ggtttcctgt cctcacagga acgaagtccc taaagaaaca
     5461 gtggcagcca ggtttagccc cggaattgac tggattcctt ttttagggcc cattggtatg
     5521 gctttttccc cgtatccccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag
     5581 aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc
     5641 ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct
     5701 agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt ccctgatatc
     5761 tataacaaga aaatatatat ataataagtt atcacgtaag tagaacatga aataacaata
     5821 taattatcgt atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc gtcattttga
     5881 ctcacgcggt cgttatagtt caaaatcagt gacacttacc gcattgacaa gcacgcctca
     5941 cgggagctcc aagcggcgac tgagatgtcc taaatgcaca gcgacggatt cgcgctattt
     6001 agaaagagag agcaatattt caagaatgca tgcgtcaatt ttacgcagac tatctttcta
     6061 gggttaatct agctgcatca ggatcatatc gtcgggtctt ttttccggct cagtcatcgc
     6121 ccaagctggc gctatctggg catcggggag gaagaagccc gtgccttttc ccgcgaggtt
     6181 gaagcggcat ggaaagagtt tgccgaggat gactgctgct gcattgacgt tgagcgaaaa
     6241 cgcacgttta ccatgatgat tcgggaaggt gtggccatgc acgcctttaa cggtgaactg
     6301 ttcgttcagg ccacctggga taccagttcg tcgcggcttt tccggacaca gttccggatg
     6361 gtcagcccga agcgcatcag caacccgaac aataccggcg acagccggaa ctgccgtgcc
     6421 ggtgtgcaga ttaatgacag cggtgcggcg ctgggatatt acgtcagcga ggacgggtat
     6481 cctggctgga tgccgcagaa atggacatgg ataccccgtg agttacccgg cgggcgcgct
     6541 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     6601 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     6661 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6721 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     6781 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     6841 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     6901 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6961 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     7021 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     7081 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     7141 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     7201 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     7261 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     7321 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     7381 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     7441 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     7501 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     7561 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     7621 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     7681 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     7741 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7801 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     7861 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     7921 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7981 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     8041 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     8101 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     8161 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     8221 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     8281 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     8341 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     8401 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     8461 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     8521 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     8581 tcctttttca atattattga agcatttatc agggt
//
