LOCUS       sequence-474413-        5691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Azenta NGS; inserts a p2A-mNeonGreen-h2b fusion at the endogenous 
            POU4F2 (aka BRN3B) gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5691)
  AUTHORS   Agarwal D, Dash N, Mazo KW, Chopra M, Avila MP, Patel A, Wong RM, 
            Jia C, Do H, Cheng J, Chiang C, Jurlina SL, Roshan M, Perry MW, Rho 
            JM, Broyer R, Lee CD, Weinreb RN, Gavrilovici C, Oesch NW, Welsbie 
            DS, Wahlin KJ
  TITLE     Human retinal ganglion cell neurons generated by synchronous BMP 
            inhibition and transcription factor mediated reprogramming.
  JOURNAL   NPJ Regen Med. 2023 Sep 29;8(1):55. doi: 10.1038/s41536-023-00327-x.
  PUBMED    37773257
REFERENCE   2  (bases 1 to 5691)
  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: "NPJ Regen 
            Med."; date: "2023-09-29"; volume: "8(1):55. doi"; pages: " 
            10.1038/s41536-023-00327-x"
COMMENT     Sequence Label: P527_POU4F2-p2A-mNeonGreen-h2b
FEATURES             Location/Qualifiers
     source          1..5691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             211..267
                     /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             268..975
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      523..532
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             976..1356
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSSSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     promoter        2422..2670
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2422..2442
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2593..2612
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2691..2766
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2867..2884)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3038..3626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3118..3137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3797..4588,4589..4657))
                     /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     4420..4439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4658..4762)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4837..4856)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    5026..5059
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             5176..5205
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5179..5208
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5182..5211
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
ORIGIN
        1 gcggagaaga agcgcaagcg cacgtccatc gctgcgccag agaagcgctc gctcgaagcc
       61 tactttgcca ttcagcctcg gccctcctct gaaaagatcg ccgccatcgc ggagaagctg
      121 gacctgaaga aaaacgtggt gcgcgtctgg ttctgcaacc agaggcagaa acagaaaaga
      181 atgaaatatt ccgccggcat tgggagcgga gctactaact tcagcctgct gaagcaggct
      241 ggagacgtgg aggagaaccc tggacctatg gtgagcaagg gcgaggagga taacatggcc
      301 tctctcccag cgacacatga gttacacatc tttggctcca tcaacggtgt ggactttgac
      361 atggtgggtc agggcaccgg caatccaaat gatggttatg aggagttaaa cctgaagtcc
      421 accaagggtg acctccagtt ctccccctgg attctggtcc ctcatatcgg gtatggcttc
      481 catcagtacc tgccctaccc tgacgggatg tcgcctttcc aggccgccat ggtagatggc
      541 tccggatacc aagtccatcg cacaatgcag tttgaagatg gtgcctccct tactgttaac
      601 taccgctaca cctacgaggg aagccacatc aaaggagagg cccaggtgaa ggggactggt
      661 ttccctgctg acggtcctgt gatgaccaac tcgctgaccg ctgcggactg gtgcaggtcg
      721 aagaagactt accccaacga caaaaccatc atcagtacct ttaagtggag ttacaccact
      781 ggaaatggca agcgctaccg gagcactgcg cggaccacct acacctttgc caagccaatg
      841 gcggctaact atctgaagaa ccagccgatg tacgtgttcc gtaagacgga gctcaagcac
      901 tccaagaccg agctcaactt caaggagtgg caaaaggcct ttaccgatgt gatgggcatg
      961 gacgagctgt acaagatgcc tgaaccctct agttctgctc cagcccctaa aaagggttct
     1021 aagaaggcta tcactaaggc gcagaagaag gatggtaaga agcgtaagcg cagccgcaag
     1081 gagagctatt ctatctatgt gtacaaggtt ctgaagcagg tccaccccga caccggcatc
     1141 tcatccaagg ccatggggat catgaactcc ttcgtcaacg acatcttcga gcgcatcgcg
     1201 ggcgaggctt ctcgcctggc tcactacaat aagcgctcga ccatcacctc cagggagatt
     1261 cagacggctg tgcgcctgct gctgcctggg gagctggcta agcatgctgt gtccgagggc
     1321 actaaggcag ttaccaagta cactagctct aaatagaaga ctcttggcct ctccagagac
     1381 gcccctttcc tcgtccgctc ttttctctcc tctcttctgc ctcttttcac ttttggcgac
     1441 tagaaacaat tccagtaaat gtgaatctcg acaaatcgag gactgaagag ggagcgaacg
     1501 agcgaacaac tgagcccaag ccggtgagaa tgtgaaacag tttctcaaag gaaagaataa
     1561 caaaagatgg tatttgtctg ttgtagcaaa gttgtccctt tgaaccccac ctcggcttct
     1621 tcagaggaag tgtggagatg gctgtttgca ggaaggcaga cgagacagtg tttaaaaagt
     1681 ccacaagaat gatcaagtaa gatttgtttt tattcttaca gacatcaccc gtgttcaagt
     1741 ttaaaagtac actttgcaac tatttttcag aaatagaaat tgattcagga ctaaaacttt
     1801 aaactagagt tgatgcttaa tgtgatagag acatctctaa agtattttga attttaaaaa
     1861 aagatggcag attttctgca tttacactgt atattatata tatattttta ttgtggttct
     1921 tacccccttt tccttctctg aagtgttaat gcttaagaaa agagttgcgc ctgctgtgtt
     1981 cactgatctt gaaagctatt attagattat tgcagaacaa ccctctgtaa attattaatt
     2041 tatctctcta gcaacttaat tttgtgcaca ttctaattaa ttaaacttct tccgtctaaa
     2101 aaaagtgggg gaaatgtata gctagtaacg ttcaaaaaat tttgtttgat gagtttaccg
     2161 aatttttaca gctttcctcc tatactgtgt tccttttgac ccatttgtat attctcactt
     2221 gaatgaagat tgtttttttc tttgttttta ctggtagtgt tctgatttgt gagtcgacac
     2281 tcagtttggt accaagcttt gcgcggtgtg gaaagtcccc aggctcccca ggcaggcaga
     2341 agtatgcaaa gcatgcatct caattagtca gcaaccagcc taggcgccta gggaattcgc
     2401 ggccgccttt tgctcacatg tgagggccta tttcccatga ttccttcata tttgcatata
     2461 cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta
     2521 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     2581 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     2641 ttatatatct tgtggaaagg acgaaacacc gccggcattt agaagactct gttttagagc
     2701 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
     2761 cggtgctttt ttggcggccg cgaccccaac tggggtaacc tttgagttct ctcagttggg
     2821 gtccataggg ataacagggt aatttgatat cgcgctcttc cgcttcctcg ctcactgact
     2881 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2941 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3001 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3061 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3121 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3181 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3241 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3301 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3361 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3421 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3481 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3541 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3601 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3661 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3721 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3781 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3841 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3901 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     3961 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4021 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4081 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4141 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4201 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4261 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4321 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4381 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4441 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4501 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4561 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4621 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4681 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4741 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     4801 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     4861 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     4921 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     4981 taggcgtttt gtgctgcttc gtagggataa cagggtaatt catgggtgcc agggcgtgcc
     5041 cttgggctcc ccgggcgcgt tcgatgtacg ggccagatat acgccaccta ccacactatg
     5101 aataccatcc cgtgcacgtc ggccgcctct tcttcatcgg tgcccatctc gcacccttcc
     5161 gcgttggcgg gcacgcacca ccaccaccac catcaccacc accaccacca ccaaccgcac
     5221 caggcgctgg agggcgagct gctggagcac ctgagtcccg ggctggccct gggcgctatg
     5281 gcgggccccg acggcgctgt ggtgtccacg ccggctcacg cgccgcacat ggccaccatg
     5341 aaccccatgc accaagcagc gctcagcatg gcccacgcgc acgggctgcc gtcgcacatg
     5401 ggctgcatga gcgacgtgga cgccgacccg cgggacctgg aggcattcgc cgagcgcttc
     5461 aagcagcgac gcatcaagct gggggtgacc caggcagatg tgggctccgc gctggccaac
     5521 ctcaagatcc ccggcgtggg ctcgcttagc cagagcacca tctgcaggtt cgagtccctc
     5581 acactgtccc acaataatat gatcgcgctc aaacccatcc tgcaggcatg gctcgaggag
     5641 gccgagaagt cccaccgcga gaagctcacc aagcctgaac tcttcaatgg c
//
