LOCUS       sequence-331140-        5447 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of Dre and TagRFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5447)
  AUTHORS   Sun D, Evans L, Perrone F, Sokleva V, Lim K, Rezakhani S, Lutolf M, 
            Zilbauer M, Rawlins EL
  TITLE     A functional genetic toolbox for human tissue-derived organoids.
  JOURNAL   eLife. 2021 Oct 6;10. pii: 67886. doi: 10.7554/eLife.67886.
  PUBMED    34612202
REFERENCE   2  (bases 1 to 5447)
  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: "eLife."; 
            date: "2021-10-6"; volume: "10. pii: 67886. doi"; pages: " 
            10.7554/eLife.67886"
COMMENT     Sequence Label: CMV-Dre-T2A-TagRFP
FEATURES             Location/Qualifiers
     source          1..5447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        402..781
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        782..985
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     936..956
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1030..1049
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1030..1048
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1084..1093
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1090..1800
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             1810..1863
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1867..1893
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2944..2964
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2989..3006)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2995..3219
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3226..3242)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3226..3242)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3239..3261)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3250..3266
                     /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(3274..3280,3281..3298,3299..3304))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3319..3340
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3457..3474)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3628..4216)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3708..3727)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4387..5178,5179..5247))
                     /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     5010..5029
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5248..5352)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5420..5438
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgacattgat tattgactag
      421 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      481 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      541 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      601 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      661 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      721 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      781 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      841 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      901 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      961 gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg cttactggct
     1021 tatcgaaatt aatacgactc actataggga gacccaagct ggctagcgtt taaacttaag
     1081 cttgccacca tggtgtctaa gggcgaagag ctgattaagg agaacatgca catgaagctg
     1141 tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag
     1201 ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc tctccccttc
     1261 gccttcgaca tcctggctac cagcttcatg tacggcagca gaaccttcat caaccacacc
     1321 cagggcatcc ccgacttctt taagcagtcc ttccctgagg gcttcacatg ggagagagtc
     1381 accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc
     1441 tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg
     1501 cagaagaaaa cactcggctg ggaggccaac accgagatgc tgtaccccgc tgacggcggc
     1561 ctggaaggca gaagcgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac
     1621 ttcaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac
     1681 tatgtggacc acagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag
     1741 cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca caaacttaat
     1801 ggatccggag agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc
     1861 cctatgtacc catacgatgt tccagattac gctggatccg gtgctagcga gctgatcatc
     1921 tctggctcct ctggaggatt cctgaggaac atcggcaagg agtaccagga ggctgctgag
     1981 aacttcatga gattcatgaa tgaccaggga gcctacgccc ctaacaccct gagagacctg
     2041 aggctggtgt tccactcctg ggctagatgg tgccacgcta gacagctggc ctggttccct
     2101 atctctcctg agatggctag ggagtacttc cttcagctgc acgatgctga cctggcctct
     2161 accaccatcg acaagcacta cgccatgctg aacatgctgc tgtcccactg tggcctgcct
     2221 cctctgtctg atgacaagtc tgtgagcctg gccatgagga gaatccggag agaggctgcc
     2281 accgagaagg gagagagaac cggccaggcc atccctctga gatgggatga cctgaagctg
     2341 ctggatgtgc tgctgtctag atctgagaga ctggtggacc tgaggaatag ggccttcctg
     2401 tttgtggcct acaacaccct gatgaggatg tctgagatct ctaggatcag agtgggagac
     2461 ctggaccaga ccggagacac cgtgaccctg cacatctccc acaccaagac catcaccacc
     2521 gctgctggcc tggacaaagt gctgtctagg aggaccaccg ctgtgctgaa tgactggctg
     2581 gatgtgtctg gcctgagaga gcaccctgac gctgtgctgt tccctcctat ccaccggagc
     2641 aacaaggcta ggatcaccac cacccctctg accgcccctg ccatggagaa gatttttagc
     2701 gatgcctggg tgctgctgaa caagagggat gccaccccta acaagggccg ctaccggacc
     2761 tggaccggcc actctgctag agtgggagct gccatcgaca tggctgagaa gcaagtgtcc
     2821 atggtggaga tcatgcagga gggcacctgg aaaaagcctg agacactgat gagatacctg
     2881 aggaggggag gagtgtctgt gggagccaac tctaggctga tggactccgc tagcggcgcc
     2941 ggtcctaaga agaagaggaa agtgtgagct ctagagctcg ctgatcagcc tcgactgtgc
     3001 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     3061 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     3121 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     3181 acaatagcag gcatgctggg gatgcggtgg gctctatggg gatccgtcat agctgtttcc
     3241 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3301 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3361 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3421 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3481 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3541 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3601 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3661 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3721 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3781 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3841 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3901 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3961 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4021 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4081 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4141 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4201 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4261 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4321 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4381 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4441 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4501 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4561 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4621 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4681 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4741 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4801 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4861 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4921 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4981 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5041 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5101 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5161 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5221 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5281 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5341 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     5401 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc
//
