LOCUS       sequence-331137-        6207 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SOX9 reporter repair template.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6207)
  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 6207)
  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: SOX9-T2A-H2B-EGFP repair template
FEATURES             Location/Qualifiers
     source          1..6207
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      17..605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     506..525
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     759..776
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    893..914
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(929..934,935..952,953..959)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    967..983
                     /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)."
     primer_bind     972..994
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     991..1007
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     991..1007
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1026..1046
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1028..1046
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1075..1091
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1076..1092
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2117..2170
                     /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             2177..2554
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      2567..2576
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2573..2575,2576..2578,2579..3292)
                     /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(2573..2575,2576..2578,2579..3289)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2618..2639)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2879..2898)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3226..3247
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4420..4439)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4421..4439)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4449..4466)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4449..4465)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4458..4480)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4606..5061)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4743..4764)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4955..4974
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5088..5192
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5193..5261,5262..6053)
                     /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     complement(5411..5430)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
       61 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      181 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      241 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      301 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      361 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      421 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      481 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      541 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      601 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      661 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      721 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      781 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      841 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
      901 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
      961 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     1021 agcgcgcaat taaccctcac taaagggaac aaaagctggg taccgggccc cccctcgagg
     1081 tcgacggtat cgataagctt ttgggcgact tatctccggt gcagcgcgcc tcttgcgcgg
     1141 gtgcgggccc ttattacact ttagcagcga gggagggtcc ccggagggtg cctaagacta
     1201 gggcgtctgc acagcccttg ttgattttct cgtgcttgtt cttttattgt ccacagggca
     1261 atcccagggc ccaccgaccc cacccaccac ccccaaaacc gacgtgcagc cgggcaaggc
     1321 tgacctgaag cgagaggggc gccccttgcc agaggggggc agacagcccc ctatcgactt
     1381 ccgcgacgtg gacatcggcg agctgagcag cgacgtcatc tccaacatcg agaccttcga
     1441 tgtcaacgag tttgaccagt acctgccgcc caacggccac ccgggggtgc cggccacgca
     1501 cggccaggtc acctacacgg gcagctacgg catcagcagc accgcggcca ccccggcgag
     1561 cgcgggccac gtgtggatgt ccaagcagca ggcgccgccg ccacccccgc agcagccccc
     1621 acaggccccg ccggccccgc aggcgccccc gcagccgcag gcggcgcccc cacagcagcc
     1681 ggcggcaccc ccgcagcagc cacaggcgca cacgctgacc acgctgagca gcgagccggg
     1741 ccagtcccag cgaacgcaca tcaagacgga gcagctgagc cccagccact acagcgagca
     1801 gcagcagcac tcgccccaac agatcgccta cagccccttc aacctcccac actacagccc
     1861 ctcctacccg cccatcaccc gctcacagta cgactacacc gaccaccaga actccagctc
     1921 ctactacagc cacgcggcag gccagggcac cggcctctac tccaccttca cctacatgaa
     1981 ccccgctcag cgccccatgt acacccccat cgccgacacc tctggggtcc cttccatccc
     2041 gcagacccac agcccccagc actgggaaca acccgtctac acacagctca ctcgacctaa
     2101 gcttgatatc gaattcgagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa
     2161 tcccggccct accggtatgc cagagccagc gaagtctgct cccgccccga aaaagggctc
     2221 caagaaggcg gtgactaagg cgcagaagaa aggcggcaag aagcgcaagc gcagccgcaa
     2281 ggagagctat tccatctatg tgtacaaggt tctgaagcag gtccaccctg acaccggcat
     2341 ttcgtccaag gccatgggca tcatgaattc gtttgtgaac gacattttcg agcgcatcgc
     2401 aggtgaggct tcccgcctgg cgcattacaa caagcgctcg accatcacct ccagggagat
     2461 ccagacggcc gtgcgcctgc tgctgcctgg ggagttggcc aagcacgccg tgtccgaggg
     2521 tactaaggcc atcaccaagt acaccagcgc taaggatcca ccggtcgcca ccatggtgag
     2581 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     2641 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2701 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2761 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2821 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2881 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2941 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     3001 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     3061 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     3121 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     3181 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3241 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aaggatccac
     3301 tagttgagga ggcctcccac gaagcgcgaa gatggccgag atgatcctaa aaataaccga
     3361 agaaagagag gaccaaccag aattcccttt ggacatttgt gtttttttgt ttttttattt
     3421 tgttttgttt tttcttcttc ttcttcttcc ttaaagacat ttaagctaaa ggcaactcgt
     3481 acccaaattt ccaagacaca aacatgacct atccaagcgc attacccact tgtggccaat
     3541 cagtggccag gccaaccttg gctaaatgga gcagcgaaat caacgagaaa ctggactttt
     3601 taaaccctct tcagagcaag cgtggaggat gatggagaat cgtgtgatca gtgtgctaaa
     3661 tctctctgcc tgtttggact ttgtaattat ttttttagca gtaattaaag aaaaaagtcc
     3721 tctgtgagga atattctcta ttttaaatat ttttagtatg tactgtgtat gattcattac
     3781 cattttgagg ggatttatac atatttttag ataaaattaa atgctcttat ttttccaaca
     3841 gctaaactac tcttagttga acagtgtgcc ctagcttttc ttgcaaccag agtatttttg
     3901 tacagatttg ctttctctta caaaaagaaa aaaaaatcct gttgtattaa catttaaaaa
     3961 cagaattgtg ttatgtgatc agttttgggg gttaactttg cttaattcct caggctttgc
     4021 gatttaagga ggagctgcct taaaaaaaaa taaaggcctt attttgcaat tatgggagta
     4081 aacaatagtc tagagaagca tttggtaagc tttatcatat atatattttt taaagaagag
     4141 aaaaacacct tgagccttaa aacggtgctg ctgggaaaca tttgcactct tttagtgcat
     4201 ttcctcctgc ctttgcttgt tcactgcagt cttaagaaag aggtaaaagg caagcaaagg
     4261 agatgaaatc tgttctggga atgtttcagc agccaataag tgcccgagca cactgccccc
     4321 ggttgcctgc ctgggcccca tgtggaaggc agatgcctgc tcgctctgtc acctgtacta
     4381 gttctagagc ggccgccacc gcggtggagc tccaattcgc cctatagtga gtcgtattac
     4441 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     4501 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     4561 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggaaatt gtaagcgtta
     4621 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     4681 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     4741 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     4801 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     4861 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     4921 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     4981 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     5041 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5101 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5161 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5221 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5281 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5341 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5401 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5461 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5521 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5581 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5641 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5701 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     5761 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     5821 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     5881 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     5941 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6001 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6061 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6121 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6181 tccactgagc gtcagacccc gtagaaa
//
