LOCUS       sequence-331127-        5638 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SOX2 CDS replacement with T2A-H2B-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5638)
  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 5638)
  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: SOX2 KO (T2A-H2B-EGFP)
FEATURES             Location/Qualifiers
     source          1..5638
                     /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"
     CDS             1440..1493
                     /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             1500..1877
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      1890..1899
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1896..1898,1899..1901,1902..2615)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1896..1898,1899..1901,1902..2612)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1941..1962)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2202..2221)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2549..2570
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3417..3433)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3417..3433)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3430..3452)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3441..3457
                     /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(3465..3471,3472..3489,3490..3495))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3510..3531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3648..3665)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3819..4407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3899..3918)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4578..5369,5370..5438))
                     /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     5201..5220
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5439..5543)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5611..5629
                     /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 ccagtaagag cgcagtacgg gagcggcacc
      421 agaggggctg gagttggggg ggagtgctgt ggatgagcgg gagaacaatg acacaccaac
      481 tcctgcactg gctgtttcca gaaatacgag ttggacagcc gccctgagcc acccactgtg
      541 ccctgcccca cccccgcacc ttagctgctt cccgcgtccc atcctcattt aagtaccctg
      601 caccaaaaag taaatcaata ttaagtttaa agaaaaaaaa acccacgtag tcttagtgct
      661 gtttacccac ttccttcgaa aaggcgtgtg gtgtgacctg ttgctgcgag aggggataca
      721 aaggtttctc agtggctggc aggctggctc tgggagcctc ctccccctcc tcgcctgccc
      781 cctcctcccc cggcctcccc cgcgcggccg gcggcgcggg aggccccgcc ccctttcatg
      841 caaaacccgg cagcgaggct gggctcgagt ggaggagccg ccgcgcgctg attggtcgct
      901 agaaacccat ttattccctg acagcccccg tcacatggat ggttgtctat taacttgttc
      961 aaaaaagtat caggagttgt caaggcagag aagagagtgt ttgcaaaagg gggaaagtag
     1021 tttgctgcct ctttaagact aggactgaga gaaagaagag gagagagaaa gaaagggaga
     1081 gaagtttgag ccccaggctt aagcctttcc aaaaaataat aataacaatc atcggcggcg
     1141 gcaggatcgg ccagaggagg agggaagcgc tttttttgat cctgattcca gtttgcctct
     1201 ctcttttttt cccccaaatt attcttcgcc tgattttcct cgcggagccc tgcgctcccg
     1261 acacccccgc ccgcctcccc tcctcctctc cccccgcccg cgggcccccc aaagtcccgg
     1321 ccgggccgag ggtcggcggc cgccggcggg ccgggcccgc gcacagcgcc cgcatgtaca
     1381 acatgatgga gacggagctg aagccgccgg gcccgcagca aacttcggcg ggatccggag
     1441 agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc cctaccggta
     1501 tgccagagcc agcgaagtct gctcccgccc cgaaaaaggg ctccaagaag gcggtgacta
     1561 aggcgcagaa gaaaggcggc aagaagcgca agcgcagccg caaggagagc tattccatct
     1621 atgtgtacaa ggttctgaag caggtccacc ctgacaccgg catttcgtcc aaggccatgg
     1681 gcatcatgaa ttcgtttgtg aacgacattt tcgagcgcat cgcaggtgag gcttcccgcc
     1741 tggcgcatta caacaagcgc tcgaccatca cctccaggga gatccagacg gccgtgcgcc
     1801 tgctgctgcc tggggagttg gccaagcacg ccgtgtccga gggtactaag gccatcacca
     1861 agtacaccag cgctaaggat ccaccggtcg ccaccatggt gagcaagggc gaggagctgt
     1921 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1981 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     2041 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     2101 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     2161 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     2221 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     2281 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     2341 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     2401 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     2461 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     2521 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     2581 ggatcactct cggcatggac gagctgtaca agtgagggcc ggacagcgaa ctggaggggg
     2641 gagaaatttt caaagaaaaa cgagggaaat gggaggggtg caaaagagga gagtaagaaa
     2701 cagcatggag aaaacccggt acgctcaaaa agaaaaagga aaaaaaaaaa tcccatcacc
     2761 cacagcaaat gacagctgca aaagagaaca ccaatcccat ccacactcac gcaaaaaccg
     2821 cgatgccgac aagaaaactt ttatgagaga gatcctggac ttctttttgg gggactattt
     2881 ttgtacagag aaaacctggg gagggtgggg agggcggggg aatggacctt gtatagatct
     2941 ggaggaaaga aagctacgaa aaacttttta aaagttctag tggtacggta ggagctttgc
     3001 aggaagtttg caaaagtctt taccaataat atttagagct agtctccaag cgacgaaaaa
     3061 aatgttttaa tatttgcaag caacttttgt acagtattta tcgagataaa catggcaatc
     3121 aaaatgtcca ttgtttataa gctgagaatt tgccaatatt tttcaaggag aggcttcttg
     3181 ctgaattttg attctgcagc tgaaatttag gacagttgca aacgtgaaaa gaagaaaatt
     3241 attcaaattt ggacatttta attgtttaaa aattgtacaa aaggaaaaaa ttagaataag
     3301 tactggcgaa ccatctctgt ggtcttgttt aaaaagggca aaagttttag actgtactaa
     3361 attttataac ttactgttaa aagcaaaaat ggccatgcag gttgacaccg ttggtagtca
     3421 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3481 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3541 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3601 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3661 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3721 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3781 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3841 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3901 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3961 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4021 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4081 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4141 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4201 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4261 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4321 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4381 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4441 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4501 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4561 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4621 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4681 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4741 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4801 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4861 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4921 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4981 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5041 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5101 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5161 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5221 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5281 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5341 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5401 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5461 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5521 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     5581 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
