LOCUS       sequence-483543-        7130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Part I of the Donor construct for UBE3A  reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7130)
  AUTHORS   Kelkar GR, Stuppy SR, Sen D, Yagci ZB, Han L, Land L, Hartman JK, 
            Keung AJ
  TITLE     A human Angelman Syndrome class II pluripotent stem cell line with 
            fluorescent paternal UBE3A reporter.
  JOURNAL   Front Cell Dev Biol. 2025 Aug 29;13:1665693. doi: 
            10.3389/fcell.2025.1665693. eCollection 2025.
  PUBMED    40950402
REFERENCE   2  (bases 1 to 7130)
  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: "2025-08-29"; volume: "13:1665693. doi"; pages: " 
            10.3389/fcell.2025.1665693. eCollection 2025"
COMMENT     Sequence Label: pSS22-IRES-NLS-eGFP
FEATURES             Location/Qualifiers
     source          1..7130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1068..1115
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             1122..1145
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1263..1281)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    1318..1439
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1329..1348)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1383..1402
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(2268..2842,2843..2845,2846..2854)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2435..2452)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2662..2681
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2858..2878
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2888..2890,2891..3604)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2888..2890,2891..3601)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2930..2951)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3191..3210)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3538..3559
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      complement(4603..5191)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4683..4702)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5272..6066)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(5272..6066)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5383..5402)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5993..6012
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        6450..6653
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6604..6624
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          6789..6921
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     6904..6923
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      6965..6974
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 aggaaggtcg tgtagccctt gagggcgaaa ctcctgtcta ccgtacctgg gtgaaatggg
       61 tcgagaccga atatcacatc aatatcttgg agacttcaga tgacgaagag ggcggttctg
      121 gaggctcagg ggggagcgga gggtccggtg gatcaggagg ctccatggca gttcccgaga
      181 cccgccctaa ccacactatt tatatcaaca acctcaatag caagatcaag aaggatgagc
      241 taaaaaagtc cctgtacgcc atcttctccc agtttggcca gatcctggat atcctggtac
      301 cgcggcagcg taccccgagg ggccaggcct ttgtcatctt caaggaggtc agcagcgcca
      361 ccaacgccct gcgctccatg cagggtttcc ctttctatga caaacctatg cgtatccagt
      421 atgccaagac cgacaaacgt atcccggcca agatgaaagg caccttcgtg ggcggtagtg
      481 gaggaagcgg ggggagcgga gggagtggtg gatccggcgg ctccatggaa ttggagatcc
      541 gtccgctttt cctggttccc gataccaatg ggttcattga ccacttagcg agcttagccc
      601 gtctgcttga gagccgtaag tacattctgg tggttcctct tattgtcatc aacgagttag
      661 acggccttgc gaaaggccag gaaacagacc atcgtgcggg aggctatgcg cgtgtcgtgc
      721 aagaaaaggc tcgcaagagc attgagtttt tggagcaacg tttcgagtcg cgtgactcct
      781 gtttacgtgc acttaccagc cgtggaaatg aacttgagtc aatcgcattt cgctctgaag
      841 atatcactgg gcagttaggt aacaatgacg atttgatttt gtcatgctgc ctgcactatt
      901 gtaaagacaa ggcaaaggac tttatgcccg cgtcgaagga agagccgatc cgtttgttgc
      961 gtgaggtggt attgcttact gatgatcgca acctgcgcgt taaggctctt acacgcaacg
     1021 tccctgtgcg tgacatccca gctttcttaa catgggccca ggtcgggaaa aggccggcgg
     1081 ccacgaaaaa ggccggccag gcaaaaaaga aaaagggatc cgattacaag gacgacgatg
     1141 acaagtagtg tttaaacgaa ttcgggctcg gtacacgggg atcctctaga gtcgacctgc
     1201 aggcatgcaa gctgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     1261 caccgctgag caataactag cataacccct tggggcggcc gcttcgagca gacatgataa
     1321 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     1381 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     1441 acaacaacaa ttgagaaaat actttgctgt cattgtgtga ggaaactttt aagaaattgc
     1501 cctagttaaa aattattatg gggctcacat tggtttggaa tcaaattagt gtgattcatt
     1561 tacttttttg attcccagct tgttaattga aagccatata acatgatcat ctatttagaa
     1621 tggttacatt gaggctcgga agattatcat ttgattgtgc tagaatcctg ttatcaaatc
     1681 attttcttag tcatattgcc agcagtgttt ctaataagca tttaagagca cacactttgc
     1741 agtcttgtaa aacaggtttg agtattttct ccaccttaga ggaagttact tgacttctca
     1801 gtgacctaac ctctaaagtg catttactga tgtcctctct gtggttttgt tgtggaaaga
     1861 tttagttaaa tgaactgtaa gaattcagta cctaaaatgg tatctgttat gtagtaaaaa
     1921 ctcaatggat acagtatctt atcatcgtca ctagctttga gtaatttata gtataaagtc
     1981 aacttgttag ttacacaaca aaaagtttat gatttgcatt aatgtatagt ttgcattgca
     2041 gaccgtctca actatataca atctaaaaat aggagcattt aattctaagt gtatttccca
     2101 tgacttacag ttttcctgtt tttttcccct tttctctatt taggttacct acatctcata
     2161 cttgctttaa tgtgctttta cttccggaat actcaagcaa agaaaaactt aaagagagat
     2221 tgttgaaggc catcacctac gctaaagact ttggcatgct ggatccgccc ctctccctcc
     2281 ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat
     2341 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct
     2401 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg
     2461 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta
     2521 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag
     2581 ccacgtgtat aagatacacc tgcaaaggcg gtacaacccc agtgccacgt tgtgagttgg
     2641 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat
     2701 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtacac atgctttaca
     2761 tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc
     2821 ctttgaaaaa cacgatgata atatggccac aaccatgcca aaaaagaaga gaaaggtaga
     2881 agaccccgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     2941 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     3001 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     3061 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     3121 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     3181 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     3241 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     3301 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     3361 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     3421 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     3481 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     3541 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     3601 gtaaaacaaa acaaaacaaa ataaaacaaa aaaaaggaag gaaaaaaaaa agaaaaaatt
     3661 taaaaaattt taaaaatata acgagggata aatttttggt ggtgatagtg tcccagtaca
     3721 aaaaggctgt aagatagtca accacagtag tcacctatgt ctgtgcctcc cttctttatt
     3781 ggggacatgt gtgctggaac agcagatttc agctacatat atgaacaaat cctttattat
     3841 tattataatt atttttttgc gtgaaagtgt tacatattct ttcacttgta tgtacagaga
     3901 ggtttttctg aatatttatt ttaagggtta aatcactttt gcttgtgttt attactgctt
     3961 gaggttgagc cttttgagta tttaaaaaat atataccaac agaactactc tcccaaggaa
     4021 aatattgcca ccatttgtag accacgtaac cttcaagtat gtgctacttt tttgtccctg
     4081 tatctaactc aaatcaggaa ctgtattttt tttaatgatt tgcttttgaa acttgaagtc
     4141 ttgaaaacag tgtgatgcaa ttactgctgt tctagccccc aaagagtttt ctgtgcaaaa
     4201 tcttgagaat caatcaataa agaaagatgg aagtaaggga gaaattggaa tgttttaact
     4261 gcagccctca gaactttagt aacagcacaa caaattaaaa acaaaaacaa ctcatgccac
     4321 agtatgtcgt cttcatgtgt cttgcaatga actgtttcag tagccaatcc tctttcttag
     4381 tatatgaaag gacagggatt tttgttcttg ttgttctcgt tgttgtttta agtttactgt
     4441 ggaaagtgca tttggccaaa tgaaatggta gtcaagccta ttgcaacaaa gttaggaagt
     4501 ttgttggcct cccttcttta ttggggacat gtggaagctt ggatcctgag caaaaggcca
     4561 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4621 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4681 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4741 gccgcttacg ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4801 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4861 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4921 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4981 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5041 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5101 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5161 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5221 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgccgt ctcagaagaa
     5281 ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag
     5341 cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa
     5401 cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa
     5461 gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc
     5521 ctcgccgtcg ggcatgctcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg
     5581 atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctct
     5641 ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag
     5701 ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag
     5761 gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac
     5821 gtcgagtaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc
     5881 gtcttgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc
     5941 ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc
     6001 atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc
     6061 aatcataata ttattgaagc atttatcagg gttcgtctcg gatccaagct tgtcactagc
     6121 tttgagtaat ttataggata aaggcaactt ggaaaagact cttcttgcag tttacaacgg
     6181 gcacagacag agcacctgtg ggaggactag gaaaattaaa gatgattata gccaaaaatg
     6241 gcccagacac agaaaggtag gtaattatta acttgtgact gtatacctac cgaaaacctt
     6301 gcattcctcg tcacatacat atgccaagtc cgccccctat tgacgtcaat gacggtaaat
     6361 ggcccgcctg gcattatgcc cagtacatga ccttacggga ctttcctact tggcagtaca
     6421 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac accaatgggc
     6481 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     6541 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaataac cccgccccgt
     6601 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
     6661 tgaaccgtca gatcactaga agctttattg cggtagttta tcacagttaa attgctaacg
     6721 cagtcagtgc ttctgacaca acagtctcga acttaagctg cagaagttgg tcgtgaggca
     6781 ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct
     6841 tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc
     6901 actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga
     6961 gtatgccacc atggatgacc catctttttt gactgggcgc agcacatacg ccaagcgtcg
     7021 ccgcgcacgc cgtatgaacg tctgcaagtg tggtgccatt cttcataaca ataaagactg
     7081 tcgcagctcc acgatctccg gccacaaact tgaccgtttg cgctttgtaa
//
