LOCUS       sequence-180697-        6323 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for generation of SOX2-ntdTomato reporter cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6323)
  AUTHORS   Balboa D, Weltner J, Novik Y, Eurola S, Wartiovaara K, Otonkoski T
  TITLE     Generation of a SOX2 reporter human induced pluripotent stem cell 
            line using CRISPR/SaCas9.
  JOURNAL   Stem Cell Res. 2017 Jul;22:16-19. doi: 10.1016/j.scr.2017.05.005. 
            Epub 2017 May 17.
  PUBMED    28952927
REFERENCE   2  (bases 1 to 6323)
  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: "2017-07"; volume: "22:16-19. doi"; pages: " 
            10.1016/j.scr.2017.05.005. Epub 2017 May 17"
COMMENT     Sequence Label: pUC19-SOX2-T2A-2xNLS-tdTomato-F2A-Puro
FEATURES             Location/Qualifiers
     source          1..6323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..29)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(183..771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(263..282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(942..1733,1734..1802))
                     /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     1565..1584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1803..1907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1975..1993
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2031..2053)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2153..2172
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2366..2388
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2380..2397
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2381..2397
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3386..3439
                     /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"
     CDS             3449..3469
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3479..3499
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3506..4933
                     /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
                     LKGEIHQALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     CDS             5000..5593
                     /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     5493..5513
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6104..6120)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6104..6120)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6117..6139)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6128..6144
                     /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)."
     promoter        complement(join(6152..6158,6159..6176,6177..6182))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6197..6218
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
       61 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      601 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
      661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1081 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1801 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1861 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1921 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1981 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     2041 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     2101 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     2161 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg
     2221 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     2281 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     2341 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa
     2401 ttggatccac agcgcccgca tgtacaacat gatggagacg gagctgaagc cgccgggccc
     2461 gcagcaaact tcggggggcg gcggcggcaa ctccaccgcg gcggcggccg gcggcaacca
     2521 gaaaaacagc ccggaccgcg tcaagcggcc catgaatgcc ttcatggtgt ggtcccgcgg
     2581 gcagcggcgc aagatggccc aggagaaccc caagatgcac aactcggaga tcagcaagcg
     2641 cctgggcgcc gagtggaaac ttttgtcgga gacggagaag cggccgttca tcgacgaggc
     2701 taagcggctg cgagcgctgc acatgaagga gcacccggat tataaatacc ggccccggcg
     2761 gaaaaccaag acgctcatga agaaggataa gtacacgctg cccggcgggc tgctggcccc
     2821 cggcggcaat agcatggcga gcggggtcgg ggtgggcgcc ggcctgggcg cgggcgtgaa
     2881 ccagcgcatg gacagttacg cgcacatgaa cggctggagc aacggcagct acagcatgat
     2941 gcaggaccag ctgggctacc cgcagcaccc gggcctcaat gcgcacggcg cagcgcagat
     3001 gcagcccatg caccgctacg acgtgagcgc cctgcagtac aactccatga ccagctcgca
     3061 gacctacatg aacggctcgc ccacctacag catgtcctac tcgcagcagg gcacccctgg
     3121 catggctctt ggctccatgg gttcggtggt caagtccgag gccagctcca gcccccctgt
     3181 ggttacctct tcctcccact ccagggcgcc ctgccaggcc ggggacctcc gggacatgat
     3241 cagcatgtat ctccccggcg ccgaggtgcc ggaacccgcc gcccccagca gacttcacat
     3301 gtcccagcac taccagagcg gcccggtgcc cggcacggcc attaacggca cactgcccct
     3361 ctcacacatg gtcgacggct ccggagaggg cagaggaagt ctgctaacat gcggtgacgt
     3421 cgaggagaat cctggcccaa tcgatggtcc aaaaaagaag agaaaggtat tcgatggtcc
     3481 aaaaaagaag agaaaggtat tcgatatggt gagcaagggc gaggaggtca tcaaagagtt
     3541 catgcgcttc aaggtgcgca tggagggctc catgaacggc cacgagttcg agatcgaggg
     3601 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     3661 cggccccctg cccttcgcct gggacatcct gtccccccag ttcatgtacg gctccaaggc
     3721 gtacgtgaag caccccgccg acatccccga ttacaagaag ctgtccttcc ccgagggctt
     3781 caagtgggag cgcgtgatga acttcgagga cggcggtctg gtgaccgtga cccaggactc
     3841 ctccctgcag gacggcacgc tgatctacaa ggtgaagatg cgcggcacca acttcccccc
     3901 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctccaccg agcgcctgta
     3961 cccccgcgac ggcgtgctga agggcgagat ccaccaggcc ctgaagctga aggacggcgg
     4021 ccactacctg gtggagttca agaccatcta catggccaag aagcccgtgc aactgcccgg
     4081 ctactactac gtggacacca agctggacat cacctcccac aacgaggact acaccatcgt
     4141 ggaacagtac gagcgctccg agggccgcca ccacctgttc ctggggcatg gcaccggcag
     4201 caccggcagc ggcagctccg gcaccgcctc ctccgaggac aacaacatgg ccgtcatcaa
     4261 agagttcatg cgcttcaagg tgcgcatgga gggctccatg aacggccacg agttcgagat
     4321 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
     4381 caagggcggc cccctgccct tcgcctggga catcctgtcc ccccagttca tgtacggctc
     4441 caaggcgtac gtgaagcacc ccgccgacat ccccgattac aagaagctgt ccttccccga
     4501 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggtctggtga ccgtgaccca
     4561 ggactcctcc ctgcaggacg gcacgctgat ctacaaggtg aagatgcgcg gcaccaactt
     4621 cccccccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct ccaccgagcg
     4681 cctgtacccc cgcgacggcg tgctgaaggg cgagatccac caggccctga agctgaagga
     4741 cggcggccgc tacctggtgg agttcaagac catctacatg gccaagaagc ccgtgcaact
     4801 gcccggctac tactacgtgg acaccaagct ggacatcacc tcccacaacg aggactacac
     4861 catcgtggaa cagtacgagc gctccgaggg ccgccaccac ctgttcctgt acggcatgga
     4921 cgagctgtac aagggatccc agctgctgaa ttttgacctt cttaagcttg cgggagacgt
     4981 cgagtccaac cctgggccca ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     5041 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     5101 caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     5161 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt
     5221 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     5281 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     5341 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca
     5401 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     5461 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     5521 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     5581 caagcccggt gccgtcgact gaattccacc cacaggcaaa tgacagctgc aaaagagaac
     5641 accaatccca tccacactca cgcaaaaacc gcgatgccga caagaaaact tttatgagag
     5701 agatcctgga cttctttttg ggggactatt tttgtacaga gaaaacctgg ggagggtggg
     5761 gagggcgggg gaatggacct tgtatagatc tggaggaaag aaagctacga aaaacttttt
     5821 aaaagttcta gtggtacggt aggagctttg caggaagttt gcaaaagtct ttaccaataa
     5881 tatttagagc tagtctccaa gcgacgaaaa aaatgtttta atatttgcaa gcaacttttg
     5941 tacagtattt atcgagataa acatggcaat caaaatgtcc attgtttata agctgagaat
     6001 ttgccaatat ttttcaagga gaggcttctt gctgaatttt gattctgcag ctgaaattta
     6061 ggacagttgc aaacgtgaaa agaagaagct tggcgtaatc atggtcatag ctgtttcctg
     6121 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     6181 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     6241 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     6301 gaggcggttt gcgtattggg cgc
//
