LOCUS       sequence-344149-        8626 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  amber suppression reporter sfGFP150TAG with EcoLeuT(CUA) amber 
            suppressor tRNA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8626)
  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 8626)
  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_4xEcoLeuT(CUA)_EF1_sfGFP150TAG
FEATURES             Location/Qualifiers
     source          1..8626
                     /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)"
     tRNA            join(1577..1610,1611..1613,1614..1660)
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     tRNA            join(1924..1957,1958..1960,1961..2007)
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     tRNA            join(2271..2304,2305..2307,2308..2354)
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     tRNA            join(2618..2651,2652..2654,2655..2701)
                     /product="amber suppressor variant of an E. coli leucine 
                     tRNA (Wu et al., 2004)"
                     /label=tRNA(CUA)
     promoter        2764..2975
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2988..3256
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      3290..3299
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(4031..4605,4606..4608,4609..4617)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4198..4215)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4425..4444
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4630..5028
                     /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(5094..5114)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5156..5277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5193..5212)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5247..5266
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6040..6074
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6565..6581)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6565..6581)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6578..6600)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6589..6605
                     /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(6613..6619,6620..6637,6638..6643))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6658..6679
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6796..6813)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6967..7555)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7047..7066)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7726..8517,8518..8586))
                     /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     8349..8368
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8587..8626,1..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 tacctcgccc ggatggtgga atctgtagac acaagggatt ctaaatccct
     1621 cggcgttcgc gctgtgcggg ttcaagtccc gctccgggta gacaagtgcg gtttttatcg
     1681 ctgcagtatt tagcatgccc cacccatctg caaggcattc tggatagtgt caaaacagcc
     1741 ggaaatcaag tccgtttatc tcaaacttta gcattttggg aataaatgat atttgctatg
     1801 ctggttaaat tagattttag ttaaatttcc tgctgaagct ctagtacgat aagtaacttg
     1861 acctaagtgt aaagttgaga tttccttcag gtttatatag cttgtgcgcc gcctgggtac
     1921 ctcgcccgga tggtggaatc ggtagacaca agggattcta aatccctcgg cgttcgcgct
     1981 gtgcgggttc aagtcccgct ccgggtagac aagtgcggtt tttcttgctg cagtatttag
     2041 catgccccac ccatctgcaa ggcattctgg atagtgtcaa aacagccgga aatcaagtcc
     2101 gtttatctca aactttagca ttttgggaat aaatgatatt tgctatgctg gttaaattag
     2161 attttagtta aatttcctgc tgaagctcta gtacgataag taacttgacc taagtgtaaa
     2221 gttgagattt ccttcaggtt tatatagctt gtgcgccgcc tgggtacctc gcccggatgg
     2281 tggaatcggt agacacaagg gattctaaat ccctcggcgt tcgcgctgtg cgggttcaag
     2341 tcccgctccg ggtagacaag tgcggttttt ttgcctgcag tatttagcat gccccaccca
     2401 tctgcaaggc attctggata gtgtcaaaac agccggaaat caagtccgtt tatctcaaac
     2461 tttagcattt tgggaataaa tgatatttgc tatgctggtt aaattagatt ttagttaaat
     2521 ttcctgctga agctctagta cgataagtaa cttgacctaa gtgtaaagtt gagatttcct
     2581 tcaggtttat atagcttgtg cgccgcctgg gtacctcgcc cggatggtgg aatcggtaga
     2641 cacaagggat tctaaatccc tcggcgttcg cgctgtgcgg gttcaagtcc cgctccgggt
     2701 agacaagtgc ggtttttact agtgctctag aaaggatctg cgatcgctcc ggtgcccgtc
     2761 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
     2821 gaacgggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     2881 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     2941 ttctttttcg caacgggttt gccgccagaa cacagctgaa gcttcgaggg gctcgcatct
     3001 ctccttcacg cgcccgccgc cctacctgag gccgccatcc acgccggttg agtcgcgttc
     3061 tgccgcctcc cgcctgtggt gcctcctgaa ctgcgtccgc cgtctaggta agtttaaagc
     3121 tcaggtcgag accgggcctt tgtccggcgc tcccttggag cctacctaga ctcagccggc
     3181 tctccacgct ttgcctgacc ctgcttgctc aactctacgt ctttgtttcg ttttctgttc
     3241 tgcgccgtta cagatccaag ctgtgaccgg cgcctactct agagctagcg ccaccatggt
     3301 gtccaagggc gaggaactgt tcaccggcgt ggtgcccatc ctggtggaac tggatggcga
     3361 cgtgaacggc cacaagttct ctgtgcgggg agagggcgaa ggcgacgcca caaatggcaa
     3421 gctgaccctg gagttcatct gcaccaccgg caagctgccc gtgccttggc ctaccctcgt
     3481 gaccacactg acctacggcg tgcagtgctt cagcagatac cccgaccata tgaagcggca
     3541 cgacttcttc aagagcgcca tgcccgaggg ctacgtgcag gaacggacca tcagcttcaa
     3601 ggacgacggc acctacaaga ccagagccga agtgaagttc gagggcgaca ccctcgtgaa
     3661 ccggatcgag ctgaagggca tcgatttcaa agaggacggc aacatcctgg gccacaagct
     3721 ggagtacaac ttcaacagcc actaggtgta catcaccgcc gacaagcaga agaacggcat
     3781 caaggccaac ttcaagatcc ggcacaacgt ggaagatggc agcgtgcagc tggccgacca
     3841 ctaccagcag aacaccccca tcggagatgg ccccgtgctg ctgcccgaca accactacct
     3901 gagcacccag agcgtgctga gcaaggaccc caacgagaag cgggaccaca tggtgctgct
     3961 ggaattcgtg accgccgctg gcatcaccca cggcatggac gagctgtaca agggcagctg
     4021 agcggccgcg cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat
     4081 aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg
     4141 tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc
     4201 tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt
     4261 cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg
     4321 acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac
     4381 cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg
     4441 tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg
     4501 ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc
     4561 cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg
     4621 gcgtccggaa tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg
     4681 gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct
     4741 agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca
     4801 gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc
     4861 gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt
     4921 ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca
     4981 gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctgacc atgggtcgac
     5041 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     5101 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgcgtt aactaaactt
     5161 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5221 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5281 tgtctggaat tgactcaaat gatgtcaatt agtctatcag aagctcatct ggtctccctt
     5341 ccgggggaca agacatccct gtttaatatt taaacagcag tgttcccaaa ctgggttctt
     5401 atatcccttg ctctggtcaa ccaggttgca gggtttcctg tcctcacagg aacgaagtcc
     5461 ctaaagaaac agtggcagcc aggtttagcc ccggaattga ctggattcct tttttagggc
     5521 ccattggtat ggctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga
     5581 gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac
     5641 gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc
     5701 tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg
     5761 tccctgatat ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg
     5821 aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg
     5881 cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca
     5941 agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat
     6001 tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga
     6061 ctatctttct agggttaatc tagctgcatc aggatcatat cgtcgggtct tttttccggc
     6121 tcagtcatcg cccaagctgg cgctatctgg gcatcgggga ggaagaagcc cgtgcctttt
     6181 cccgcgaggt tgaagcggca tggaaagagt ttgccgagga tgactgctgc tgcattgacg
     6241 ttgagcgaaa acgcacgttt accatgatga ttcgggaagg tgtggccatg cacgccttta
     6301 acggtgaact gttcgttcag gccacctggg ataccagttc gtcgcggctt ttccggacac
     6361 agttccggat ggtcagcccg aagcgcatca gcaacccgaa caataccggc gacagccgga
     6421 actgccgtgc cggtgtgcag attaatgaca gcggtgcggc gctgggatat tacgtcagcg
     6481 aggacgggta tcctggctgg atgccgcaga aatggacatg gataccccgt gagttacccg
     6541 gcgggcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     6601 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     6661 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6721 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6781 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     6841 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     6901 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6961 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     7021 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     7081 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     7141 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     7201 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     7261 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     7321 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     7381 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     7441 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     7501 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     7561 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     7621 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     7681 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     7741 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     7801 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     7861 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     7921 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     7981 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     8041 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     8101 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     8161 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     8221 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     8281 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     8341 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     8401 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     8461 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     8521 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     8581 actcatactc ttcctttttc aatattattg aagcatttat cagggt
//
