LOCUS       sequence-474418-        5091 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used in CRISPR gene editing to Insert a p2A-h2b_mRuby3 sequence 
            before the stop codon of the endogenous human VSX2 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5091)
  AUTHORS   Jones MK, Agarwal D, Mazo KW, Chopra M, Jurlina SL, Dash N, Xu Q, 
            Ogata AR, Chow M, Hill AD, Kambli NK, Xu G, Sasik R, Birmingham A, 
            Fisch KM, Weinreb RN, Enke RA, Skowronska-Krawczyk D, Wahlin KJ
  TITLE     Chromatin Accessibility and Transcriptional Differences in Human 
            Stem Cell-Derived Early-Stage Retinal Organoids.
  JOURNAL   Cells. 2022 Oct 28;11(21):3412. doi: 10.3390/cells11213412.
  PUBMED    36359808
REFERENCE   2  (bases 1 to 5091)
  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: "Cells."; 
            date: "2022-10-28"; volume: "11(21):3412. doi"; pages: " 
            10.3390/cells11213412"
COMMENT     Sequence Label: P521_VSX2_p2A-h2b_mRuby3
FEATURES             Location/Qualifiers
     source          1..5091
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             113..169
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             170..547
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSSSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     CDS             563..1270
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTDI
                     ALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAVA
                     KYSNLGGGMDELYK"
     promoter        1945..2193
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1945..1965
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2116..2135
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2214..2289
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2390..2407)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2561..3149)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2641..2660)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3320..4111,4112..4180))
                     /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     3943..3962
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4181..4285)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4360..4379)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    4549..4581
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
ORIGIN
        1 ggacagcctg gcccggagta ccgagaagcc agaggaggag gaggccatgg atgaagacag
       61 gccggcggag aggctcagtc caccgcagct ggaggacatg gctggaagtg gagctactaa
      121 cttcagccta cttaagcagg ctggagacgt ggaggagaac cctggaccca tgcctgaacc
      181 ctctagttct gctccagccc ctaaaaaggg ttctaagaag gctatcacta aggcgcagaa
      241 gaaggatggt aagaagcgta agcgcagccg caaggagagc tattctatct atgtgtacaa
      301 ggttctgaag caggtccacc ccgacaccgg catctcatcc aaggccatgg ggatcatgaa
      361 ctccttcgtc aacgacatct tcgagcgcat cgcgggcgag gcttctcgcc tggctcacta
      421 caataagcgc tcgaccatca cctccaggga gattcagacg gctgtgcgcc tgctgctgcc
      481 tggggagctg gctaagcatg ctgtgtccga gggcactaag gcagttacca agtacactag
      541 ctctaaggat ccaccggtcg ccgtgtctaa gggcgaagag ctgatcaagg aaaatatgcg
      601 tatgaaggtg gtcatggaag gttcggtcaa cggccaccaa ttcaaatgca caggtgaagg
      661 agaaggcaga ccgtacgagg gagtgcaaac catgaggatc aaagtcatcg agggaggacc
      721 cctgccattt gcctttgaca ttcttgccac gtcgttcatg tatggcagcc gtacctttat
      781 caagtacccg gccgacatcc ctgatttctt taaacagtcc tttcctgagg gttttacttg
      841 ggaaagagtt acgagatacg aagatggtgg agtcgtcacc gtcacgcagg acaccagcct
      901 tgaggatggc gagctcgtct acaacgtcaa ggtcagaggg gtaaactttc cctccaatgg
      961 tcccgtgatg cagaagaaga ccaagggttg ggagcctaat acagagatga tgtatccagc
     1021 agatggtggt ctgagaggat acactgacat cgcactgaaa gttgatggtg gtggccatct
     1081 gcactgcaac ttcgtgacaa cttacaggtc aaaaaagacc gtcgggaaca tcaagatgcc
     1141 cggtgtccat gccgttgatc accgcctgga aaggatcgag gagagtgaca atgaaaccta
     1201 cgtagtgcaa agagaagtgg cagttgccaa atacagcaac cttggtggtg gcatggacga
     1261 gctgtacaag taagtcaagg cgcgctcaga tgccggagcc ccaagactct gctctcctcg
     1321 ggccctgtgg tgctgggaga tgctctctga ggcaaggccc agacctggcc tctgccatcc
     1381 tccctgttcc ccacaggtcc tccatcaccc ctggtggctg caggcaccgc tgggttctga
     1441 ctctggacca tgctgagaca tccctcatct agtcttgacc tctccagcat cccagcctca
     1501 gaagccttct tgctgcccac aacgtcccct caatcccctt ctctcaatcc ctttgcaacg
     1561 ctcactggtt ttggccaccc cttgctctct gttctcttgc tttaaagagt cctccttccc
     1621 agctctacat tctgctctgc ccatgcctaa agcccattgc tgcaaatgca ttgtgaattg
     1681 cctgccatgg ctgtgacaca gacggaggac tggaactgca actccagcgt cctcagcacc
     1741 ccactcctca gtaaaagtct tctcccaact cagcctgttc cttcctgcag acctggctgg
     1801 gcctgggctt ccacagtgtg aagacactgt gcaggcccat ttggtaccaa gctttgcgcg
     1861 gatgtatgtg tgtgcgtgta cgtgtgtatg tgaagatacc gcctaggcgc ctagggaatt
     1921 cgcggccgcc ttttgctcac atgtgagggc ctatttccca tgattccttc atatttgcat
     1981 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
     2041 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     2101 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     2161 gctttatata tcttgtggaa aggacgaaac accggaggac atggcttagg tcagttttag
     2221 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     2281 agtcggtgct tttttggcgg ccgcgacccc aactggggta acctttgagt tctctcagtt
     2341 ggggtccata gggataacag ggtaatttga tatcgcgctc ttccgcttcc tcgctcactg
     2401 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2461 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2521 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2581 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2641 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2701 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2761 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2821 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     2881 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     2941 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3001 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3061 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3121 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3181 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3241 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3301 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3361 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3421 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3481 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3541 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3601 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3661 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3721 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3781 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     3841 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     3901 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     3961 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4021 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4081 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4141 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4201 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4261 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     4321 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     4381 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     4441 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
     4501 ggttaggcgt tttgcgctgc ttcgtaggga taacagggta attcatgggt gccagggcgt
     4561 gcccttgggc tccccgggcg cttcgatgta cgggccagat atagcatgtg ctgtggcctg
     4621 gaagggacag aacaggccac ccgaggccca ggtgcccagg gctttggcag ggggaggtcc
     4681 tccacagggc tggcgaccca tctccccatt ccctgaccct ggtccagccc tgggacttgt
     4741 gtgactgcgg tgtggggagt aaggctttct gctcgtcctt aattctggcc tctctctatc
     4801 tttgccgttt tcagttcaag atggctttcc caggcgcttt tctaaacccg aataccaaca
     4861 attctttcta gggatgcaca aaaagtcgct ggaggcagca gccgagtcgg ggaggaagcc
     4921 cgagggggaa cgccaggccc tgcccaagct cgacaagatg gagcaggacg agcggggccc
     4981 cgacgctcag gcggccatct cccaggagga actgagggag aacagcattg cggtgctccg
     5041 ggccaaagct caggagcaca gcaccaaagt gctggggact gtgtctgggc c
//
