LOCUS       sequence-220877-        7143 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology arms and linker-mEGFP sequence for C-terminus tagging of 
            human TNNI1, via excisable Cas9-excisable hPGK-mCherry selection 
            cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7143)
  AUTHORS   Roberts B, Hendershott MC, Arakaki J, Gerbin KA, Malik H, Nelson A, 
            Gehring J, Hookway C, Ludmann SA, Yang R, Haupt A, Grancharova T, 
            Valencia V, Fuqua MA, Tucker A, Rafelski SM, Gunawardane RN
  TITLE     Fluorescent Gene Tagging of Transcriptionally Silent Genes in 
            hiPSCs.
  JOURNAL   Stem Cell Reports. 2019 May 14;12(5):1145-1158. doi: 
            10.1016/j.stemcr.2019.03.001. Epub 2019 Apr 4.
  PUBMED    30956114
REFERENCE   2  (bases 1 to 7143)
  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.stemcr.2019.03.001"; journalName: "Stem Cell Reports"; 
            date: "2019-05-14- 14"; volume: "12"; issue: "5"; pages: "1145-1158"
COMMENT     Sequence Label: AICSDP-34: TNNI1-mEGFP
FEATURES             Location/Qualifiers
     source          1..7143
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    217..338
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(254..273)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     308..327
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        355..855
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     774..792
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     primer_bind     complement(1022..1040)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1290..1309
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1595..1819
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             join(1888..1890,1891..2604)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1930..1951)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2191..2210)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2538..2559
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3636..3652)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3636..3652)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3649..3671)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3660..3676
                     /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(3684..3690,3691..3708,3709..3714))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3729..3750
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3867..3884)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4038..4626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4118..4137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4797..5588,5589..5657))
                     /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     5420..5439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5658..5762)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5830..5848
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5886..5908)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6008..6027
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6221..6243
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6235..6252
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6236..6252
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gatgtgtgtg ccacttgtct acaggagcgg cctgtggagg tgggtgactg gaggaagaac
       61 gtggaggcca tgtctggcat ggaaggccgg aagaagatgt ttgatgccgc caagtctccg
      121 acctcacaat ccgggtccgg gtcccccgga gaggttcccg acataccaat aacttcgtat
      181 agtgtaggat atacgaagtt atattagtat tccgataact tgtttattgc agcttataat
      241 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
      301 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgatt ggggttgcgc
      361 cttttccaag gcagccctgg gtttgcgcag ggacgcggct gctctgggcg tggttccggg
      421 aaacgcagcg gcgccgaccc tgggtctcgc acattcttca cgtccgttcg cagcgtcacc
      481 cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt
      541 cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc
      601 actagtaccc tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg accgcgatgg
      661 gctgtggcca atagcggctg ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg
      721 gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc
      781 gcattctgca agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc
      841 gacctctctc cccaggggga tccaccggag cttaccatgg tctccaaagg agaagaagat
      901 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
      961 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     1021 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
     1081 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     1141 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     1201 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     1261 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
     1321 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     1381 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     1441 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc
     1501 cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc
     1561 ggcggaatgg atgaactcta taaatgaggt actcctgtgc cttctagttg ccagccatct
     1621 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1681 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     1741 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     1801 gatgcggtgg gctctatgga aataacttcg tatagtgtag gatatacgaa gttatatagg
     1861 tatgtcggga acctctccgg gtccggggtg agcaagggcg aggagctgtt caccggggtg
     1921 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1981 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     2041 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     2101 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     2161 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     2221 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     2281 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     2341 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     2401 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     2461 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccaagctgag caaagacccc
     2521 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     2581 ggcatggacg agctgtacaa gtagaggtac ctagagacca gctcccttct ctggggtcca
     2641 tcagagtcta gaggataatg agggagtcca caatgggcag gcgggttgta cttagtgtgg
     2701 tgcacggatg cacggactaa ggcaccgatg agactgattt gagtcggctc tcctgggctc
     2761 agtcctggct ccagcaccat gatacttggg catgtcattt ctgtttggcg gccctttttg
     2821 ttcccattat ctatgaaatt gggggctgta tcaaatcagt ggttctccaa gtgtggtcct
     2881 gggaccagca gcgtctacat cacctgggat gcaaatgatc agcccccacc ccagatccac
     2941 taaattggaa actctggggt gaggccagcc ttctaggtga ttctgatgca catgagagtt
     3001 tgagaaccac taggttagag ggtccctggg atcccttcat ctctgacttt ctgagaatct
     3061 atgcactttt aaatacctcc tggtgatgtc ctttatttgt gctccctgcg aagtacccag
     3121 cactcacagt acctgttccc aaggagctac cactcacaga agctaagact tcccaccatc
     3181 tgaggaaata gctggagaaa ctgggcaaag caaggaaatc aggccacgta aaaccctcta
     3241 cggaagtgct gagagaatgt gcaatagggc aggcatccac cagacagcac tggtcagagc
     3301 aggctttctg gagaggagga gctctctgag gatgcgacga gcgagtgtta gctgataaag
     3361 ggaagggcat gttgaagggc tgatggtgca acagaagcat ggaggtggga attaagcagc
     3421 cacagagtag catggcaagg ctaggtaaga gctgagggcc gcagtgtgga ggagcggtgt
     3481 gaggggtccc aaaaggggag caactgtgcc cgagggccga gaagctgagg ttttgtagga
     3541 gcccttatga atgaagatcc agtccggggt gcgctagggc tgagaagccc tggggactgc
     3601 aatcccaatg gcgcgccgag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3661 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3721 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3781 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3841 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3901 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3961 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4021 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4081 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4141 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4201 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4261 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4321 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4381 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4441 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4501 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4561 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4621 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4681 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4741 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4801 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4861 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4921 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4981 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5041 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5101 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5161 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5221 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5281 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5341 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5401 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5461 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5521 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5581 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5641 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5701 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5761 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     5821 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     5881 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     5941 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     6001 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     6061 cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac
     6121 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     6181 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     6241 acgacggcca gtgaattgac gcgtattggg atcaccctct gccctaccag ctttcccctt
     6301 gtcaacccct cacatggcct caggctggcc caggagctcc tgttggagac cagtcagctt
     6361 gacagacaga acattctaga atgctttggg agaccagtgt gggaagaaag aaagaagatt
     6421 tgctcccttc cttcaggctg ttcccagtct gacgaagaaa ctaggacata aagaaagcta
     6481 gaccccagac ggagaaatga agtaaaaatc ttgttcccag tgtgaagata atgtggagat
     6541 ttagggtaat ggtggagatg actgcctggt caccacaggg aaaggagaca gcagctcatc
     6601 ttggttatag ccacacacga gcatagacat tagagaactg agttcaacca cccagcaaca
     6661 aattattcaa agttggttga attcacagta tatgtgcccc aaattgatag gctctgtaag
     6721 gaacagggat gaaggaacag tctctggccc aagaagcttc agaggagaag agataagcca
     6781 gacaatatac tgaggaaatg gaaaagcagc cgcatttgca ggggaagact tcttggaaga
     6841 ggtggccttg agcctgctga gtgccagacc aggagggtgg catggagcct tggaggaggt
     6901 tttgctgagg tgcactgagg tgggaactag ccccatagct gccactttca cccagtgctc
     6961 ccaagccaca cagcttcctt tttcctgtgg ggcctcggct tggcctgtgt ccacctgcct
     7021 ggctgagtcc taggacctta ccagctgggg aggggcatca ggatttctta tcatccgagg
     7081 agactggttt cctatcaccc agcttctctt gagctggggg gccgtccagc ctggtaagtc
     7141 cct
//
