LOCUS       sequence-344143-        8630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  amber suppression reporter sfGFP150TAG expression, with BstTyr(CUA) 
            amber suppressor tRNA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8630)
  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 8630)
  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_4xBstTyrT(CUA)_EF1 sfGFP150TAG
FEATURES             Location/Qualifiers
     source          1..8630
                     /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        2768..2979
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2992..3260
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      3294..3303
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(4035..4609,4610..4612,4613..4621)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4202..4219)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4429..4448
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4634..5032
                     /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(5098..5118)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5160..5281
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5197..5216)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5251..5270
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6044..6078
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6569..6585)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6569..6585)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6582..6604)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6593..6609
                     /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(6617..6623,6624..6641,6642..6647))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6662..6683
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6800..6817)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6971..7559)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7051..7070)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7730..8521,8522..8590))
                     /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     8353..8372
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8591..8630,1..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 agtggccttg gagctgcagt atttagcatg ccccacccat ctgcaaggca ttctggatag
     1381 tgtcaaaaca gccggaaatc aagtccgttt atctcaaact ttagcatttt gggaataaat
     1441 gatatttgct atgctggtta aattagattt tagttaaatt tcctgctgaa gctctagtac
     1501 gataagtaac ttgacctaag tgtaaagttg agatttcctt caggtttata tagcttgtgc
     1561 gccgcctggg tacctcggag gggtagcgaa gtggctaaac gcggcggact ctaaatccgc
     1621 tccctttggg ttcggcggtt cgaatccgtc cccctccaga caagtgcggt ttttatcgct
     1681 gcagtattta gcatgcccca cccatctgca aggcattctg gatagtgtca aaacagccgg
     1741 aaatcaagtc cgtttatctc aaactttagc attttgggaa taaatgatat ttgctatgct
     1801 ggttaaatta gattttagtt aaatttcctg ctgaagctct agtacgataa gtaacttgac
     1861 ctaagtgtaa agttgagatt tccttcaggt ttatatagct tgtgcgccgc ctgggtacct
     1921 cggaggggta gcgaagtggc taaacgcggc ggactctaaa tccgctccct ttgggttcgg
     1981 cggttcgaat ccgtccccct ccagacaagt gcggtttttc atgctgcagt atttagcatg
     2041 ccccacccat ctgcaaggca ttctggatag tgtcaaaaca gccggaaatc aagtccgttt
     2101 atctcaaact ttagcatttt gggaataaat gatatttgct atgctggtta aattagattt
     2161 tagttaaatt tcctgctgaa gctctagtac gataagtaac ttgacctaag tgtaaagttg
     2221 agatttcctt caggtttata tagcttgtgc gccgcctggg tacctcggag gggtagcgaa
     2281 gtggctaaac gcggcggact ctaaatccgc tccctttggg ttcggcggtt cgaatccgtc
     2341 cccctccaga caagtgcggt ttttactagt ggccttggag ctgcagtatt tagcatgccc
     2401 cacccatctg caaggcattc tggatagtgt caaaacagcc ggaaatcaag tccgtttatc
     2461 tcaaacttta gcattttggg aataaatgat atttgctatg ctggttaaat tagattttag
     2521 ttaaatttcc tgctgaagct ctagtacgat aagtaacttg acctaagtgt aaagttgaga
     2581 tttccttcag gtttatatag cttgtgcgcc gcctgggtac ctcggagggg tagcgaagtg
     2641 gctaaacgcg gcggactcta aatccgctcc ctttgggttc ggcggttcga atccgtcccc
     2701 ctccagacaa gtgcggtttt tactagtgct ctagaaagga tctgcgatcg ctccggtgcc
     2761 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
     2821 aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
     2881 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
     2941 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg aggggctcgc
     3001 atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg gttgagtcgc
     3061 gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta ggtaagttta
     3121 aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc tagactcagc
     3181 cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt ttcgttttct
     3241 gttctgcgcc gttacagatc caagctgtga ccggcgccta ctctagagct agcgccacca
     3301 tggtgtccaa gggcgaggaa ctgttcaccg gcgtggtgcc catcctggtg gaactggatg
     3361 gcgacgtgaa cggccacaag ttctctgtgc ggggagaggg cgaaggcgac gccacaaatg
     3421 gcaagctgac cctggagttc atctgcacca ccggcaagct gcccgtgcct tggcctaccc
     3481 tcgtgaccac actgacctac ggcgtgcagt gcttcagcag ataccccgac catatgaagc
     3541 ggcacgactt cttcaagagc gccatgcccg agggctacgt gcaggaacgg accatcagct
     3601 tcaaggacga cggcacctac aagaccagag ccgaagtgaa gttcgagggc gacaccctcg
     3661 tgaaccggat cgagctgaag ggcatcgatt tcaaagagga cggcaacatc ctgggccaca
     3721 agctggagta caacttcaac agccactagg tgtacatcac cgccgacaag cagaagaacg
     3781 gcatcaaggc caacttcaag atccggcaca acgtggaaga tggcagcgtg cagctggccg
     3841 accactacca gcagaacacc cccatcggag atggccccgt gctgctgccc gacaaccact
     3901 acctgagcac ccagagcgtg ctgagcaagg accccaacga gaagcgggac cacatggtgc
     3961 tgctggaatt cgtgaccgcc gctggcatca cccacggcat ggacgagctg tacaagggca
     4021 gctgagcggc cgcgcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg
     4081 gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc
     4141 aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc
     4201 cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa
     4261 gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct
     4321 ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca
     4381 caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca
     4441 agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat
     4501 ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc
     4561 cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac
     4621 catggcgtcc ggaatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc
     4681 aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct
     4741 ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg
     4801 tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat
     4861 cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt
     4921 gcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac
     4981 ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct gaccatgggt
     5041 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     5101 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg cgttaactaa
     5161 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     5221 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     5281 atcatgtctg gaattgactc aaatgatgtc aattagtcta tcagaagctc atctggtctc
     5341 ccttccgggg gacaagacat ccctgtttaa tatttaaaca gcagtgttcc caaactgggt
     5401 tcttatatcc cttgctctgg tcaaccaggt tgcagggttt cctgtcctca caggaacgaa
     5461 gtccctaaag aaacagtggc agccaggttt agccccggaa ttgactggat tcctttttta
     5521 gggcccattg gtatggcttt ttccccgtat ccccccaggt gtctgcaggc tcaaagagca
     5581 gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc
     5641 gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc cccgggcggc
     5701 tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct ttgggggggg
     5761 gctgtccctg atatctataa caagaaaata tatatataat aagttatcac gtaagtagaa
     5821 catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt aaaagataat
     5881 catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt
     5941 gacaagcacg cctcacggga gctccaagcg gcgactgaga tgtcctaaat gcacagcgac
     6001 ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt caattttacg
     6061 cagactatct ttctagggtt aatctagctg catcaggatc atatcgtcgg gtcttttttc
     6121 cggctcagtc atcgcccaag ctggcgctat ctgggcatcg gggaggaaga agcccgtgcc
     6181 ttttcccgcg aggttgaagc ggcatggaaa gagtttgccg aggatgactg ctgctgcatt
     6241 gacgttgagc gaaaacgcac gtttaccatg atgattcggg aaggtgtggc catgcacgcc
     6301 tttaacggtg aactgttcgt tcaggccacc tgggatacca gttcgtcgcg gcttttccgg
     6361 acacagttcc ggatggtcag cccgaagcgc atcagcaacc cgaacaatac cggcgacagc
     6421 cggaactgcc gtgccggtgt gcagattaat gacagcggtg cggcgctggg atattacgtc
     6481 agcgaggacg ggtatcctgg ctggatgccg cagaaatgga catggatacc ccgtgagtta
     6541 cccggcgggc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     6601 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     6661 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     6721 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6781 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     6841 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6901 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6961 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     7021 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     7081 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     7141 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     7201 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     7261 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     7321 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     7381 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     7441 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     7501 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     7561 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     7621 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     7681 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     7741 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     7801 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     7861 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     7921 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7981 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     8041 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     8101 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     8161 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     8221 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     8281 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     8341 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     8401 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     8461 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     8521 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     8581 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
//
