LOCUS       sequence-107390-        7295 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing P2A_NLS_sfGFP with homologous arm of Nanog..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7295)
  AUTHORS   Yu C, Liu Y, Ma T, Liu K, Xu S, Zhang Y, Liu H, La Russa M, Xie M, 
            Ding S, Qi LS
  TITLE     Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem 
            Cells.
  JOURNAL   Cell Stem Cell. 2015 Feb 5;16(2):142-7. doi: 
            10.1016/j.stem.2015.01.003.
  PUBMED    25658371
REFERENCE   2  (bases 1 to 7295)
  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"; doi: 
            "10.1016/j.stem.2015.01"; journalName: "Cell Stem Cell"; date: 
            "2015-02-5- 5"; volume: "16"; issue: "2"; pages: "142-7"
COMMENT     Sequence Label: pP2A_NLS_sfGFP
FEATURES             Location/Qualifiers
     source          1..7295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(49..66)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      219..265
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(318..335)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(489..1077)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(569..588)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1248..2039,2040..2108))
                     /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     1871..1890
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2109..2213)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3978..4034
                     /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             4041..4067
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4047..4073
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             4086..4106
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4113..4133
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4173..4886
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             4173..4883
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(4200..4228)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4823..4843
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
ORIGIN
        1 aaacggtctc cagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat
       61 taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt
      121 ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt
      181 ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt
      241 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaactggatc cttactcgag
      301 tctagactgc aggcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
      361 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
      421 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
      481 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
      541 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
      601 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
      661 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
      721 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
      781 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
      841 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
      901 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
      961 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     1021 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     1081 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     1141 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     1201 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     1261 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     1321 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     1381 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     1441 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     1501 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     1561 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     1621 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     1681 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     1741 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     1801 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     1861 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     1921 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     1981 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     2041 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     2101 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     2161 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     2221 ccccgaaaag tgccacctga caagttgctg taatgtaaca caatgagact gacttgctaa
     2281 tgaatcgaac taacgtctgg acgtctctct aactatccta tcctcctatc ctatgcgcct
     2341 gttaaaatca taattatgtg attgattgat tgattgatta tgtataagta cactatagct
     2401 gtcttcagat acaccagaag agggcatcag atctcattac ggatggttgt gagccaccat
     2461 gtggttgctg ggatttgaac tcaggacctt cggaagagca gtcagtgctc ttaaccgctg
     2521 agccatctct ccagccccaa ttatgtgatt tatatatttc ccagtttgtt tcattggtta
     2581 ctttatcttc tacttcacct cccacaccca tccccaaatc agggtctcac tgtagccctg
     2641 actggtctct gtagaccagg ctgggagagg ttcacctgcc tctgtctcct gggtgctgga
     2701 aataaaggtg tgcctggctt ttttcctttc ttttcttttt taagattgtt tatttttaaa
     2761 ttcctttagt tcattggtgt tttgcctgca tgtgtttgtc tttgtgagag tatcagatct
     2821 cctggactag agttacagac agtcgtgagc tgccatgtgg ccgctgagaa ttgaacccag
     2881 gtcctctgga agagcagcca gtgctcttaa cagaagagcc atttccccca gcacctgtct
     2941 ttttcaagaa agagtttcac tttacaggcc tggctgtcat gtttaaggtc gggctgtaga
     3001 cctgtctgca ttctgtatgt cttagcttgt tcgaatgtcc tgtcactctg caggttaaga
     3061 cctggtttca aaaccaaagg atgaagtgca agcggtggca gaaaaaccag tggttgaaga
     3121 ctagcaatgg tctgattcag gtaacaagat gttttctctg ctttcctttt tccccctcca
     3181 tcttaccatc ttattttatg tgtctgtgcc gtgtgtgtgt gtgtgtgtgt gtgtgtggct
     3241 gacctatggc tgttgggtat ggatcccaga tttcctgtaa gagcagtcag tggctctgcc
     3301 tttgcttccc agtgtcatcc tgatccatct atgtccatta gtcctgtccc tttcccatcc
     3361 ctcttcctcc acgcactctt tcttgtgatc tttggttctt tgaggcaggc tctctctatg
     3421 tagccctggc agtcagagat ctgcctgcct agtctcagga gtgctggggt taacggcctg
     3481 tgcggccatg ctaatctttt cagtatttct gcagtttaca tctttagagt ccccttttca
     3541 ctcattgcat taatcacctc tttcagaagg gctcagcacc agtggagtat cccagcatcc
     3601 attgcagcta tccccagggc tatctggtga acgcatctgg aagcctttcc atgtggggca
     3661 gccagacttg gaccaaccca acttggagca gccagacctg gaccaaccca acttggaaca
     3721 accagacctg gaccaaccca acttggagca gccaggcctg gaccgctcag tcctggaacg
     3781 gccagccttg gaatgctgct ccgctccata acttcgggga ggactttctg cagccttacg
     3841 tacagttgca gcaaaacttc tctgccagtg atttggaggt gaatttggaa gccactaggg
     3901 aaagccatgc gcattttagc accccacaag ccttggaatt attcctgaac tactctgtga
     3961 ctccaccagg tgaaatagct actaacttca gcctgctgaa gcaggctgga gacgtggagg
     4021 agaaccctgg acctatggcc tatccctatg acgtgcccga ttatgccagc ctgggcagcg
     4081 gctcccccaa gaaaaaacgc aaggtggaag atcctaagaa aaagcggaaa gtggacggca
     4141 ttggtagtgg gagcaacggc agcagcggat ccagcaaagg agaagaactt ttcactggag
     4201 ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtccgtg
     4261 gagagggtga aggtgatgct acaaacggaa aactcaccct taaatttatt tgcactactg
     4321 gaaaactacc tgttccgtgg ccaacacttg tcactactct gacctatggt gttcaatgct
     4381 tttcccgtta tccggatcac atgaaacggc atgacttttt caagagtgcc atgcccgaag
     4441 gttatgtaca ggaacgcact atatctttca aagatgacgg gacctacaag acgcgtgctg
     4501 aagtcaagtt tgaaggtgat acccttgtta atcgtatcga gttaaagggt attgatttta
     4561 aagaagatgg aaacattctt ggacacaaac tcgagtacaa ctttaactca cacaatgtat
     4621 acatcacggc agacaaacaa aagaatggaa tcaaagctaa cttcaaaatt cgccacaacg
     4681 ttgaagatgg ttccgttcaa ctagcagacc attatcaaca aaatactcca attggcgatg
     4741 gccctgtcct tttaccagac aaccattacc tgtcgacaca atctgtcctt tcgaaagatc
     4801 ccaacgaaaa gcgtgaccac atggtccttc ttgagtttgt aactgctgct gggattacac
     4861 atggcatgga tgagctctac aaatgagact tacgcaacat ctgggcttaa agtcagggca
     4921 aagccaggtt ccttccttct tccaaatatt ttcatatttt ttttaaagat ttatttattc
     4981 attatatgta agtacactgt agctgtcttc agacactcca gaagagggcg tcagatcttg
     5041 ttacgtatgg ttgtgagcca ccatgtggtt gctgggattt gaactcctga ccttcggaag
     5101 agcagtcggg tgctcttatc cactgagcca tctcaccagc ccctggttta tttttttaat
     5161 tattatttgc tttttgttta tcaagacagg gtttctctgc atagctctaa ttgtctttga
     5221 actagctctg cagaccagcc tggccttgaa ctcagagatc tgcccactta tctttgcctc
     5281 ctgaatgctg ggaccaaagg tggcatacca ccacacctgg catatatatt gtttatttct
     5341 atttctattt ttattggtgc cagagcaaac ctaggactta gaacatgctg ggcaccaact
     5401 caacttctga gctctattta caacttggtg tgttagtgta tttgtcttag ttctgaattt
     5461 gtcctttttt tagtgttaac tctaggcttt ggagacagtg aggtgcatat actctctcct
     5521 tcccaagaat aagtgcttga acacccttac ccacgcccac ccacccatgc tagtcttttt
     5581 tcttagaagc gtgggtcttg gtatacactg tgtcattttg aggggtgagg tttaaaagta
     5641 tatacaaagt ataacgatat ggtggctact ctcgaggatg agacagaagg accaggagtt
     5701 tgagggtagc tcagatatgc aataagttca aggccaacct gtactatgtt taaatagtaa
     5761 gacagcatct cgataaaata ataaaactaa agtctcaaca aaataaaagc tttcacctat
     5821 taaggtgctt gcttgtcctt ggagtccccc aagagtaact gctatgttaa tatctgtaga
     5881 aagatgttta tatttgactg taccatgatg aaccgatgcc agctggacta gtttaaacaa
     5941 aataaaacac taattttacc tttagtggtc ttatcttttg tccagtgggg tcaatatact
     6001 ttttattttg gtatgtgaag gggagggtct taaccgaggg tccaggaaaa tgggtttctt
     6061 ttggcttaac cggttgggag ggttatacca caacagttgg aatggcctgg taaccagagc
     6121 aggaggccag ccactcacaa tgcatccagt caagaactgg agagcaatga ataagaaatg
     6181 aatgttttag tgtgggtatg ggccctgagg aggccagaag cattcgatga tcccctggag
     6241 atgaagttac gtgaagccac ctgatcacct gatgtggcag gctgcaggga aataagctct
     6301 agttctctgg aagaacaaat atttaactgg gagagtggtt tttattcctg tgaaataata
     6361 gaacttgtgg agagagcccc tgaggtcaca cacctagtgc ttctgggaaa gaggagggac
     6421 cacagtctct tgtttttgtg tcacactgga gattaatctg ggacttcctg gctccagatg
     6481 gaggctatct tccctgagca taacccccta acctacgtgg gggtggggga agatgtcaag
     6541 tgttatcaca ggagacttga gaggctgtgt gggttcagaa gtgagtttgg ggttttggag
     6601 acgatttaaa tttgctgaaa agacagaaga tacgtataca tctggtgtag ttgtgtgtac
     6661 acttaaggag attaagttaa cgtaactcct gatgcattaa ttatagatgt tatagtagtt
     6721 gtatacagta gatttagcta aagcatgata cggaaaaaca cagaagaagc aagcgacacc
     6781 tagtaactgc cgtgcgtaaa tagccatacc tgtcaatcac atctttaact cctgaccact
     6841 cacaggggta cccagcccag cccaagtagg ttctgtagct gctcctcagc taaggagagg
     6901 gctgttttgg cagtgagaaa aattgtccct cattgtgcca cagaggctag gacacattcc
     6961 cttgcaaagc ccagactaat tccacctaga attctcctct tctgctttct gtcctttggc
     7021 atcagtgggg atggctgctg tgtcagagat gggacatcat cacctccaca aaccctgacg
     7081 accgttactg agagaccact ttgcttcaac tcttagaggt ttcgttgata agattctaaa
     7141 atgtgtggtc ttggggatca aatccaggga ctcttagacc ctgggtaagt gctctactac
     7201 tgaacaacac tcagtccttg gtgtgagaat ccgagctttg attttaccct ttttagggtg
     7261 gaagagattc caccaatcac ccaggagtgg ggcta
//
