LOCUS       sequence-474475-        6556 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human donor plasmid for integration of p2A-h2b-EGFP into the 3' end 
            of POU4F2 (aka BRN3B). This includes the donor casette and a U6 
            driven guide sequence to be used with Cas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6556)
  AUTHORS   Wahlin KJ, Cheng J, Jurlina SL, Jones MK, Dash NR, Ogata A, Kibria 
            N, Ray S, Eldred KC, Kim C, Heng JS, Phillips J, Johnston RJ Jr, 
            Gamm DM, Berlinicke C, Zack DJ
  TITLE     CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of 
            Brain and Optic Transcriptional Differences in Human PSC-Derived 
            Organoids.
  JOURNAL   Front Cell Dev Biol. 2021 Nov 16;9:764725. doi: 
            10.3389/fcell.2021.764725. eCollection 2021.
  PUBMED    34869356
REFERENCE   2  (bases 1 to 6556)
  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: "Front Cell 
            Dev Biol."; date: "2021-11-16"; volume: "9:764725. doi"; pages: " 
            10.3389/fcell.2021.764725. eCollection 2021"
COMMENT     Sequence Label: P532_POU4F2-p2A-tdTomato-p2A-Thy1.2
FEATURES             Location/Qualifiers
     source          1..6556
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             47..535
                     /codon_start=1
                     /product="mouse GPI-anchored cell surface glycoprotein of 
                     the immunoglobulin superfamily"
                     /label=Thy1.2
                     /note="This allele of Thy1 is also known as CD90.2."
                     /translation="MNPAISVALLLSVLQVSRGQKVTSLTACLVNQNLRLDCRHENNTK
                     DNSIQHEFSLTREKRKHVLSGTLGIPEHTYRSRVTLSNQPYIKVLTLANFTTKDEGDYF
                     CELQVSGANPMSSNKSISVYRDKLVKCGGISLLVQNTSWMLLLLLSLSLLQALDFISL"
     promoter        1572..1820
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1572..1592
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1743..1762
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1841..1916
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2017..2034)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2188..2776)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2268..2287)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2947..3738,3739..3807))
                     /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     3570..3589
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3808..3912)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3987..4006)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    4176..4209
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             4326..4355
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4329..4358
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4332..4361
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5052..5108
                     /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             5109..6536
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     CDS             join(6546..6556,1..46)
                     /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"
ORIGIN
        1 cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctatga acccagccat
       61 cagcgtcgct ctcctgctct cagtcttgca ggtgtcccga gggcagaagg tgaccagcct
      121 gacagcctgc ctggtgaacc aaaaccttcg cctggactgc cgccatgaga ataacaccaa
      181 ggataactcc atccagcatg agttcagcct gacccgagag aagaggaagc acgtgctctc
      241 aggcaccctt gggatacccg agcacacgta ccgctcccgc gtcaccctct ccaaccagcc
      301 ctatatcaag gtccttaccc tagccaactt caccaccaag gatgagggcg actacttttg
      361 tgagcttcaa gtctcgggcg cgaatcccat gagctccaat aaaagtatca gtgtgtatag
      421 agacaagctg gtcaagtgtg gcggcataag cctgctggtt cagaacacat cctggatgct
      481 gctgctgctg ctttccctct ccctcctcca agccctggac ttcatttctc tgtgatagaa
      541 gactcttggc ctctccagag acgccccttt cctcgtccgc tcttttctct cctctcttct
      601 gcctcttttc acttttggcg actagaaaca attccagtaa atgtgaatct cgacaaatcg
      661 aggactgaag agggagcgaa cgagcgaaca actgagccca agccggtgag aatgtgaaac
      721 agtttctcaa aggaaagaat aacaaaagat ggtatttgtc tgttgtagca aagttgtccc
      781 tttgaacccc acctcggctt cttcagagga agtgtggaga tggctgtttg caggaaggca
      841 gacgagacag tgtttaaaaa gtccacaaga atgatcaagt aagatttgtt tttattctta
      901 cagacatcac ccgtgttcaa gtttaaaagt acactttgca actatttttc agaaatagaa
      961 attgattcag gactaaaact ttaaactaga gttgatgctt aatgtgatag agacatctct
     1021 aaagtatttt gaattttaaa aaaagatggc agattttctg catttacact gtatattata
     1081 tatatatttt tattgtggtt cttaccccct tttccttctc tgaagtgtta atgcttaaga
     1141 aaagagttgc gcctgctgtg ttcactgatc ttgaaagcta ttattagatt attgcagaac
     1201 aaccctctgt aaattattaa tttatctctc tagcaactta attttgtgca cattctaatt
     1261 aattaaactt cttccgtcta aaaaaagtgg gggaaatgta tagctagtaa cgttcaaaaa
     1321 attttgtttg atgagtttac cgaattttta cagctttcct cctatactgt gttccttttg
     1381 acccatttgt atattctcac ttgaatgaag attgtttttt tctttgtttt tactggtagt
     1441 gttctgattt gtgagtcgac actcagtttg gtaccaagct ttgcgcggat gtatgtgtgt
     1501 gcgtgtacgt gtgtatgtga agataccgcc taggcgccta gggaattcgc ggccgccttt
     1561 tgctcacatg tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     1621 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     1681 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     1741 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     1801 tgtggaaagg acgaaacacc gccggcattt agaagactct gttttagagc tagaaatagc
     1861 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     1921 ttggcggccg cgaccccaac tggggtaacc tttgagttct ctcagttggg gtccataggg
     1981 ataacagggt aatttgatat cgcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     2041 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2101 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     2161 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     2221 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     2281 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2341 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     2401 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     2461 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     2521 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     2581 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     2641 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     2701 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     2761 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     2821 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     2881 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     2941 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3001 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3061 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     3121 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3181 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3241 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     3301 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3361 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     3421 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     3481 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     3541 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     3601 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     3661 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     3721 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     3781 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     3841 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3901 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
     3961 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4021 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
     4081 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
     4141 gtgctgcttc gtagggataa cagggtaatt catgggtgcc agggcgtgcc cttgggctcc
     4201 ccgggcgcgt tcgatgtacg ggccagatat acgccaccta ccacactatg aataccatcc
     4261 cgtgcacgtc ggccgcctct tcttcatcgg tgcccatctc gcacccttcc gcgttggcgg
     4321 gcacgcacca ccaccaccac catcaccacc accaccacca ccaaccgcac caggcgctgg
     4381 agggcgagct gctggagcac ctgagtcccg ggctggccct gggcgctatg gcgggccccg
     4441 acggcgctgt ggtgtccacg ccggctcacg cgccgcacat ggccaccatg aaccccatgc
     4501 accaagcagc gctcagcatg gcccacgcgc acgggctgcc gtcgcacatg ggctgcatga
     4561 gcgacgtgga cgccgacccg cgggacctgg aggcattcgc cgagcgcttc aagcagcgac
     4621 gcatcaagct gggggtgacc caggcagatg tgggctccgc gctggccaac ctcaagatcc
     4681 ccggcgtggg ctcgcttagc cagagcacca tctgcaggtt cgagtccctc acactgtccc
     4741 acaataatat gatcgcgctc aaacccatcc tgcaggcatg gctcgaggag gccgagaagt
     4801 cccaccgcga gaagctcacc aagcctgaac tcttcaatgg cgcggagaag aagcgcaagc
     4861 gcacgtccat cgctgcgcca gagaagcgct cgctcgaagc ctactttgcc attcagcctc
     4921 ggccctcctc tgaaaagatc gccgccatcg cggagaagct ggacctgaag aaaaacgtgg
     4981 tgcgcgtctg gttctgcaac cagaggcaga aacagaaaag aatgaaatat tccgccggca
     5041 ttgggagcgg agctactaac ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc
     5101 ctggacccat ggtgagcaag ggcgaggagg tcatcaaaga gttcatgcgc ttcaaggtgc
     5161 gcatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     5221 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg
     5281 cctgggacat cctgtccccc cagttcatgt acggctccaa ggcgtacgtg aagcaccccg
     5341 ccgacatccc cgattacaag aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     5401 tgaacttcga ggacggcggt ctggtgaccg tgacccagga ctcctccctg caggacggca
     5461 cgctgatcta caaggtgaag atgcgcggca ccaacttccc ccccgacggc cccgtaatgc
     5521 agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc
     5581 tgaagggcga gatccaccag gccctgaagc tgaaggacgg cggccactac ctggtggagt
     5641 tcaagaccat ctacatggcc aagaagcccg tgcaactgcc cggctactac tacgtggaca
     5701 ccaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgct
     5761 ccgagggccg ccaccacctg ttcctggggc atggcaccgg cagcaccggc agcggcagct
     5821 ccggcaccgc ctcctccgag gacaacaaca tggccgtcat caaagagttc atgcgcttca
     5881 aggtgcgcat ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg
     5941 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc
     6001 ccttcgcctg ggacatcctg tccccccagt tcatgtacgg ctccaaggcg tacgtgaagc
     6061 accccgccga catccccgat tacaagaagc tgtccttccc cgagggcttc aagtgggagc
     6121 gcgtgatgaa cttcgaggac ggcggtctgg tgaccgtgac ccaggactcc tccctgcagg
     6181 acggcacgct gatctacaag gtgaagatgc gcggcaccaa cttccccccc gacggccccg
     6241 taatgcagaa gaagaccatg ggctgggagg cctccaccga gcgcctgtac ccccgcgacg
     6301 gcgtgctgaa gggcgagatc caccaggccc tgaagctgaa ggacggcggc cactacctgg
     6361 tggagttcaa gaccatctac atggccaaga agcccgtgca actgcccggc tactactacg
     6421 tggacaccaa gctggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     6481 agcgctccga gggccgccac cacctgttcc tgtacggcat ggacgagctg tacaagggga
     6541 gcggagctac taactt
//
