LOCUS       sequence-180693-        4895 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for generation of OCT4-T2A-nEmGFP reporter cell 
            lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4895)
  AUTHORS   Balboa D, Weltner J, Novik Y, Eurola S, Wartiovaara K, Otonkoski T
  TITLE     Generation of an OCT4 reporter human induced pluripotent stem cell 
            line using CRISPR/SpCas9.
  JOURNAL   Stem Cell Res. 2017 Aug;23:105-108. doi: 10.1016/j.scr.2017.07.006. 
            Epub 2017 Jul 11.
  PUBMED    28925359
REFERENCE   2  (bases 1 to 4895)
  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: "Stem Cell 
            Res."; date: "2017-08"; volume: "23:105-108. doi"; pages: " 
            10.1016/j.scr.2017.07.006. Epub 2017 Jul 11"
COMMENT     Sequence Label: pUC19-OCT4-T2A-NLS-EmGFP-P2A-Puro
FEATURES             Location/Qualifiers
     source          1..4895
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     178..197
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     437..458
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(510..530)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(540..560)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(570..623)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(921..938)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(921..937)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(930..952)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1146..1165)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1265..1287
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1325..1343)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1411..1515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1516..1584,1585..2376)
                     /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     complement(1734..1753)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2547..3135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3036..3055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3289..3306
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3423..3444
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3459..3464,3465..3482,3483..3489)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3497..3513
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3502..3524
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3521..3537
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3521..3537
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(4022..4615)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4102..4122)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             complement(4616..4672)
                     /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             complement(join(4682..4895,1..497,498..500,501..503))
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4724..4745)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 gtcttgaagt tcaccttgat gccgttcttc tgcttgtcgg cggtgatata gaccttgtgg
       61 ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
      121 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
      181 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg
      241 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcgggcgaa gcactgcacg
      301 ccgtaggtga aggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
      361 atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg
      421 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
      481 agctcctcgc ccttgctcac catatcgaat acctttctct tcttttttgg accatcgaat
      541 acctttctct tcttttttgg accatcgatt gggccaggat tctcctcgac gtcaccgcat
      601 gttagcagac ttcctctgcc ctctccggag ccgtcgacgt ttgaatgcat gggagagccc
      661 agagtggtga cggagacagg gggaaaggct tccccctcag ggaaagggac cgaggagtac
      721 agtgcagtga agtgagggct cccatagcct ggggtaccaa aatggggccc tggggccaga
      781 ggaaaggaca ctggtccccc tgagaaagga gacccagcag cctcaaaatc ctctcgttgt
      841 gcatagtcgc tgcttgatcg cttgcccttc tggcgccggt tacagaacca cactcggacc
      901 acatccttct cgagcaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
      961 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     1021 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     1081 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
     1141 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     1201 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     1261 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     1321 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     1381 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1441 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     1501 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     1561 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     1621 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     1681 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     1741 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     1801 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     1861 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     1921 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     1981 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2041 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2101 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2161 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2221 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2281 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2341 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     2401 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     2461 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2521 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2581 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2641 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     2701 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2761 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2821 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2881 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2941 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3001 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3061 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3121 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3181 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3241 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3301 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     3361 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     3421 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     3481 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     3541 gattacgcca agcttccaga gtagggtggg aaagcaaaaa gccagctctg aacaggtaac
     3601 agctacatgg tgactgagtc tatgggcaaa agttcttgca tcacaggctt ttgggaacta
     3661 gcctatcaca gggccctgta caaataaact tggctgcaat cccagctctc cctctgatgt
     3721 tgtgtgacct taaggagtgt aaatggcacc ttagtttcag ggtcacttgg gtatgagcat
     3781 tggatattcc catccccacc tcagtaactg aaggacaaac caagataagt gtgtctatct
     3841 actgtgtccc aagcttcttt atttaagaaa aaagtgatac atgatgtggg attaaaatca
     3901 agagcatcat tgaacttcac cttccctcca accagttgcc ccaaactccc ctgcccccac
     3961 cctttgtgtt cccaattcct tccttagtga atgaagaacg tcgacttcga attcagtcga
     4021 cggcaccggg cttgcgggtc atgcaccagg tgcgcggtcc ttcgggcacc tcgacgtcgg
     4081 cggtgacggt gaagccgagc cgctcgtaga aggggaggtt gcggggcgcg gaggtctcca
     4141 ggaaggcggg caccccggcg cgctcggccg cctccactcc ggggagcacg acggcgctgc
     4201 ccagaccctt gccctggtgg tcgggcgaga ctccgacggt ggccaggaac cacgcgggct
     4261 ccttgggccg gtgcggcgcc aggaggcctt ccatctgttg ctgcgcggcc agccgggaac
     4321 cgctcaactc ggccatgcgc gggccgatct cggcgaacac cgcccccgct tcgacgctct
     4381 ccggcgtggt ccagaccgcc accgcggcgc cgtcgtccgc gacccacacc ttgccgatgt
     4441 cgagcccgac gcgcgtgagg aagagttctt gcagctcggt gacccgctcg atgtggcggt
     4501 ccggatcgac ggtgtggcgc gtggcggggt agtcggcgaa cgcggcggcg agggtgcgta
     4561 cggccctggg gacgtcgtcg cgggtggcga ggcgcaccgt gggcttgtac tcggtcggtc
     4621 caggattctc ttcgacatct ccggcttgtt tcagcagaga gaagtttgtt gcgccggatc
     4681 ccttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga
     4741 ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt
     4801 ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg
     4861 cgagctgcac gctgccgtcc tcgatgttgt ggcgg
//
