LOCUS       sequence-344604-        8549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  opal suppression reporter sfGFP150TGA expression, with MmaPylT(UCA) 
            opal suppressor tRNA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8549)
  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 8549)
  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_4xMmaPylT(UCA)_EF1_sfGFP150TGA
FEATURES             Location/Qualifiers
     source          1..8549
                     /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        2687..2898
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2911..3179
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      3213..3222
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(3954..4528,4529..4531,4532..4540)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4121..4138)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4348..4367
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4553..4951
                     /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(5017..5037)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5079..5200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5116..5135)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5170..5189
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5963..5997
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6488..6504)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6488..6504)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6501..6523)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6512..6528
                     /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(6536..6542,6543..6560,6561..6566))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6581..6602
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6719..6736)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6890..7478)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6970..6989)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7649..8440,8441..8509))
                     /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     8272..8291
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8510..8549,1..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 agttctgcag tatttagcat gccccaccca tctgcaaggc attctggata gtgtcaaaac
     1381 agccggaaat caagtccgtt tatctcaaac tttagcattt tgggaataaa tgatatttgc
     1441 tatgctggtt aaattagatt ttagttaaat ttcctgctga agctctagta cgataagtaa
     1501 cttgacctaa gtgtaaagtt gagatttcct tcaggtttat atagcttgtg cgccgcctgg
     1561 gtacctcgga aacctgatca tgtagatcga acggacttca aatccgttca gccgggttag
     1621 attcccgggg tttccggaca agtgcggttt ttcctgcagt atttagcatg ccccacccat
     1681 ctgcaaggca ttctggatag tgtcaaaaca gccggaaatc aagtccgttt atctcaaact
     1741 ttagcatttt gggaataaat gatatttgct atgctggtta aattagattt tagttaaatt
     1801 tcctgctgaa gctctagtac gataagtaac ttgacctaag tgtaaagttg agatttcctt
     1861 caggtttata tagcttgtgc gccgcctggg tacctcggaa acctgatcat gtagatcgaa
     1921 cggacttcaa atccgttcag ccgggttaga ttcccggggt ttccggacaa gtgcggtttt
     1981 tcctgcagta tttagcatgc cccacccatc tgcaaggcat tctggatagt gtcaaaacag
     2041 ccggaaatca agtccgttta tctcaaactt tagcattttg ggaataaatg atatttgcta
     2101 tgctggttaa attagatttt agttaaattt cctgctgaag ctctagtacg ataagtaact
     2161 tgacctaagt gtaaagttga gatttccttc aggtttatat agcttgtgcg ccgcctgggt
     2221 acctcggaaa cctgatcatg tagatcgaac ggacttcaaa tccgttcagc cgggttagat
     2281 tcccggggtt tccggacaag tgcggttttt cctgcagtat ttagcatgcc ccacccatct
     2341 gcaaggcatt ctggatagtg tcaaaacagc cggaaatcaa gtccgtttat ctcaaacttt
     2401 agcattttgg gaataaatga tatttgctat gctggttaaa ttagatttta gttaaatttc
     2461 ctgctgaagc tctagtacga taagtaactt gacctaagtg taaagttgag atttccttca
     2521 ggtttatata gcttgtgcgc cgcctgggta cctcggaaac ctgatcatgt agatcgaacg
     2581 gacttcaaat ccgttcagcc gggttagatt cccggggttt ccggacaagt gcggtttttc
     2641 actagtgctc tagaaaggat ctgcgatcgc tccggtgccc gtcagtgggc agagcgcaca
     2701 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg tgcctagaga
     2761 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     2821 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     2881 tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc acgcgcccgc
     2941 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt
     3001 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc
     3061 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg
     3121 accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg ttacagatcc
     3181 aagctgtgac cggcgcctac tctagagcta gcgccaccat ggtgtccaag ggcgaggaac
     3241 tgttcaccgg cgtggtgccc atcctggtgg aactggatgg cgacgtgaac ggccacaagt
     3301 tctctgtgcg gggagagggc gaaggcgacg ccacaaatgg caagctgacc ctgaagttca
     3361 tctgcaccac cggcaagctg cccgtgcctt ggcctaccct cgtgaccaca ctgacctacg
     3421 gcgtgcagtg cttcagcaga taccccgacc atatgaagcg gcacgacttc ttcaagagcg
     3481 ccatgcccga gggctacgtg caggaacgga ccatcagctt caaggacgac ggcacctaca
     3541 agaccagagc cgaagtgaag ttcgagggcg acaccctcgt gaaccggatc gagctgaagg
     3601 gcatcgattt caaagaggac ggcaacatcc tgggccacaa gctggagtac aacttcaaca
     3661 gccactgagt gtacatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     3721 tccggcacaa cgtggaagat ggcagcgtgc agctggccga ccactaccag cagaacaccc
     3781 ccatcggaga tggccccgtg ctgctgcccg acaaccacta cctgagcacc cagagcgtgc
     3841 tgagcaagga ccccaacgag aagcgggacc acatggtgct gctggaattc gtgaccgccg
     3901 ctggcatcac ccacggcatg gacgagctgt acaagggcag ctaagcggcc gcgcccctct
     3961 ccctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt
     4021 gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct
     4081 ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa
     4141 ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg
     4201 tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc
     4261 caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg
     4321 agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg
     4381 aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc
     4441 tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg
     4501 gttttccttt gaaaaacacg atgataatat ggccacaacc atggcgtccg gaatggccaa
     4561 gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat
     4621 ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt
     4681 cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg
     4741 cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa
     4801 caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg
     4861 gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt
     4921 gctgccctct ggttatgtgt gggagggctg accatgggtc gacaatcaac ctctggatta
     4981 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     5041 atacgctgct ttaatgcctt tgtatcatgc gttaactaaa cttgtttatt gcagcttata
     5101 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5161 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg aattgactca
     5221 aatgatgtca attagtctat cagaagctca tctggtctcc cttccggggg acaagacatc
     5281 cctgtttaat atttaaacag cagtgttccc aaactgggtt cttatatccc ttgctctggt
     5341 caaccaggtt gcagggtttc ctgtcctcac aggaacgaag tccctaaaga aacagtggca
     5401 gccaggttta gccccggaat tgactggatt ccttttttag ggcccattgg tatggctttt
     5461 tccccgtatc cccccaggtg tctgcaggct caaagagcag cgagaagcgt tcagaggaaa
     5521 gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg cacgctgccg gctcggggat
     5581 gcggggggag cgccggaccg gagcggagcc ccgggcggct cgctgctgcc ccctagcggg
     5641 ggagggacgt aattacatcc ctgggggctt tggggggggg ctgtccctga tatctataac
     5701 aagaaaatat atatataata agttatcacg taagtagaac atgaaataac aatataatta
     5761 tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt ttgactcacg
     5821 cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc ctcacgggag
     5881 ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct atttagaaag
     5941 agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt tctagggtta
     6001 atctagctgc atcaggatca tatcgtcggg tcttttttcc ggctcagtca tcgcccaagc
     6061 tggcgctatc tgggcatcgg ggaggaagaa gcccgtgcct tttcccgcga ggttgaagcg
     6121 gcatggaaag agtttgccga ggatgactgc tgctgcattg acgttgagcg aaaacgcacg
     6181 tttaccatga tgattcggga aggtgtggcc atgcacgcct ttaacggtga actgttcgtt
     6241 caggccacct gggataccag ttcgtcgcgg cttttccgga cacagttccg gatggtcagc
     6301 ccgaagcgca tcagcaaccc gaacaatacc ggcgacagcc ggaactgccg tgccggtgtg
     6361 cagattaatg acagcggtgc ggcgctggga tattacgtca gcgaggacgg gtatcctggc
     6421 tggatgccgc agaaatggac atggataccc cgtgagttac ccggcgggcg cgcttggcgt
     6481 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     6541 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     6601 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     6661 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     6721 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     6781 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     6841 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6901 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6961 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     7021 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     7081 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     7141 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     7201 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     7261 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     7321 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     7381 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     7441 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     7501 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     7561 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     7621 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     7681 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     7741 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     7801 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     7861 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7921 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7981 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     8041 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     8101 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     8161 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     8221 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     8281 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     8341 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     8401 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     8461 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     8521 ttcaatatta ttgaagcatt tatcagggt
//
