LOCUS       sequence-474411-        5310 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human donor plasmid for integration of p2A-h2b-EGFP into the 3' end 
            of SIX6. 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 5310)
  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 5310)
  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: P530_SIX6-p2A-h2b-EGFP
FEATURES             Location/Qualifiers
     source          1..5310
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             43..99
                     /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             100..477
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSSSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     CDS             join(493..495,496..1209)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(535..556)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(796..815)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1143..1164
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        1963..2211
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1963..1983
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2134..2153
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2232..2307
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2408..2425)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2579..3167)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2659..2678)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3338..4129,4130..4198))
                     /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     3961..3980
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4199..4303)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4378..4397)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    4567..4600
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
ORIGIN
        1 tccatcacgt ccagcgacag cgagtgcgac atcggaagtg gagctactaa cttcagccta
       61 cttaagcagg ctggagacgt ggaagagaac cctggaccta tgcctgaacc ctctagttct
      121 gctccagccc ctaaaaaggg ttctaagaag gctatcacta aggcgcagaa gaaggatggt
      181 aagaagcgta agcgcagccg caaggagagc tattctatct atgtgtacaa ggttctgaag
      241 caggtccacc ccgacaccgg catctcatcc aaggccatgg ggatcatgaa ctccttcgtc
      301 aacgacatct tcgagcgcat cgcgggcgag gcttctcgcc tggctcacta caataagcgc
      361 tcgaccatca cctccaggga gattcagacg gctgtgcgcc tgctgctgcc tggggagctg
      421 gctaagcatg ctgtgtccga gggcactaag gcagttacca agtacactag ctctaaggat
      481 ccaccggtcg ccgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
      541 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
      601 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
      661 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
      721 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
      781 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
      841 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
      901 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
      961 cagaagaacg gcatcaaggc gaacttcaag atccgccaca acatcgagga cggcggcgtg
     1021 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     1081 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     1141 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     1201 tacaagtaag ttgctcatcc atgatgctca gaagcagatt ccagtgtaaa aacgagaaaa
     1261 acaaaatgaa agaggggaag aagatgagag acctgcaaat ccagcgccag agaagccagg
     1321 tgaccaggga cccgcgggct cgggttgccg tttcccgccc caccccgcgg ccggcctggc
     1381 ttcactggcg ccctttggcc gcgaccacgg gaaccagcgg tgaggcctga cccagcacca
     1441 cgttcttctt gctttgcttt ttcctaagga ttttgctgca aagtctcctt cggaacccga
     1501 actgcaagct gagcgcctgc ccagattctc ccatgggtat ttcacgtcga aaggacgctg
     1561 ttacatatgt ataactttcg ctttaaagtt tttttttaac aaaacatata tatgctgttt
     1621 atttacttat ttaagagacc gccatggtag gtttctctgt agcttgggga acttgctgtt
     1681 tctaaacatg caggctggtg gtgatgggtt ctgtgtggag aagccaaaca ataaaacaac
     1741 ctagtgggca accttcttaa ttaagggagc tgctctcaga ttccttttct tcttattatt
     1801 attaatatca ttcccttcac caggcatgca gggacccttg agcaaatggt ctccggattt
     1861 ggtaccaagc tttgcgcgga tgtatgtgtg tgcgtgtacg tgtgtatgtg aagataccgc
     1921 ctaggcgcct agggaattcg cggccgcctt ttgctcacat gtgagggcct atttcccatg
     1981 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
     2041 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     2101 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     2161 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgacatctga
     2221 gttgcccatc cgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     2281 cttgaaaaag tggcaccgag tcggtgcttt tttggcggcc gcgaccccaa ctggggtaac
     2341 ctttgagttc tctcagttgg ggtccatagg gataacaggg taatttgata tcgcgctctt
     2401 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2461 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2521 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgtgttg ctggcgtttt
     2581 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2641 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2701 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2761 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2821 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2881 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2941 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3001 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3061 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3121 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3181 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3241 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3301 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3361 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3421 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     3481 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3541 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3601 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3661 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3721 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     3781 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     3841 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     3901 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3961 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4021 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4081 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4141 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4201 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4261 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4321 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     4381 aatctgctct gatgccgcat agttaagcca gtatctactc cctgcttgtg tgttggaggt
     4441 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     4501 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgtagggata acagggtaat
     4561 tcatgggtgc cagggcgtgc ccttgggctc cccgggcgcg ttcgatgtac gggccagata
     4621 tatttcgttt cccggagtgt ggatctcgat cagccaaaca ttttgcggaa gagcccggcc
     4681 tcatccccca ggcccaaatg ctccttacaa tcctttttgc ctttaggtcg ggccgacccg
     4741 atccaacgcg atcgcgggag cacttgctca ggcgtaagcc ccaggcagac gcaccgttag
     4801 aaatggtatc ccatgtccct gggaccgatc tgtccttgtc acccacgctt cgtttatttc
     4861 ctgacagtcc tgtaaatctc ccaaaagtgc acaacaaaca gggaggacac tgcaagccca
     4921 gtatataaaa gacctgggag ctgcggcgct gagaaagggc gcgaatcatg gtggggcaca
     4981 acagtaggga cccgcggagg ggcggccgcg gactcctgcc cgacctctgt cgccttgccg
     5041 agtaatcctc gccttaactg ctggggtctt cggaagaacc tctagccgcc gggctggagg
     5101 gacgcaggag gtggtggggg cgggcgacgg gcggctgtgt tacgagctgt gacccgtgtt
     5161 ccctttcttc cccgtagact ccagcagcag gtcctgtcac agggttccgg gcgggcacta
     5221 cgggcggagg gcgacggcac gccagaggtg ctgggcgtcg ccaccagccc ggccgccagt
     5281 ctatccagca aggcggccac ttcagccatc
//
