LOCUS       sequence-220885-        7861 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology arms and linker-mEGFP sequence for C-terminus tagging of 
            human TTN, via Cas9-excisable CAGGS-mCherry selection cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7861)
  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 7861)
  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-45: TTN-mEGFP
FEATURES             Location/Qualifiers
     source          1..7861
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1088..1209
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1125..1144)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1179..1198
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     enhancer        1224..1603
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1884..2901
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2825..2847
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2909..2928
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(3129..3147)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3397..3416
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3702..3926
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             join(3995..3997,3998..4711)
                     /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(4037..4058)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4298..4317)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4645..4666
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(join(5749..5755,5756..5773,5774..5779))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5794..5815
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5932..5949)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6103..6691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6183..6202)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6862..7653,7654..7722))
                     /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     7485..7504
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7723..7827)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 swawmyrwyt aatcattttt ctcctagtga ttcctagcac ctgcatgtaa tgtaatgata
       61 gaaatataag gattcatgtt ttggagacat tgagctagct cttgacaaat gtatgtatat
      121 atgtatttgc ttatgcttca acagcaaatt gtttgggtta tgctgctgtg tgtgtgttga
      181 agtttccatt tctgtaagtc agcgtcatat gtcatcaatt aaccagatgt cattgatgtt
      241 cactgtgtat ctgctatgct ccacagctct agttgaagaa ccttccagag aggtagtatt
      301 gagaacaagt ggtgacacaa gcttgcaagg aagcttctcg tctcagtcag tccaaatgtc
      361 tgcctccaag caggaggcct ccttcagcag tttcagcagc agcagtgcta gcagcatgac
      421 tgagatgaaa tttgcaagca tgtctgccca aagcatgtcc tccatgcaag agtcctttgt
      481 agaaatgagt tccagcagct ttatgggaat atctaatatg acacaactgg aaagctcaac
      541 tagtaaaatg cttaaagcag gcataagagg tgagcaataa aattactggt agaggtagac
      601 aagccaagta acatggtgta gtctttagta cacacaattc agtagaaagc aaatctttat
      661 ttttttcagt gattctaaga cctttttttt gtcattgttc cataaaggaa ttccgcctaa
      721 aattgaagct cttccatctg atatcagcat tgatgaaggc aaagttctaa cagtagcctg
      781 tgctttcacg ggtgagccta ccccagaagt aacatggtcc tgtggtggaa gaaaaatcca
      841 cagtcaagaa caggggaggt tccacattga aaacacagat gacctgacaa ccctgatcat
      901 catggacgta cagaaacaag atggtggact ttataccctg agtttaggga atgaatttgg
      961 atctgactct gcaactgtga atatacatat tcgatccatt tccgggtccg ggtcccccgg
     1021 agaggttccc gacataccaa taacttcgta tagtgtagga tatacgaagt tatattagta
     1081 ttccgataac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     1141 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     1201 tgtatcttat catgtctggt ctcgacattg attattgact agttattaat agtaatcaat
     1261 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     1321 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     1381 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     1441 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     1501 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     1561 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
     1621 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
     1681 tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccgggggg
     1741 gggggggggg gggggggggg ggggsggggs grggsggrga ggkgcggcgg cagccaatca
     1801 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
     1861 aaagcgaagc gcgcggcggg cggggagtcg ctgcgacgct gccttcgccc cgtgccccgc
     1921 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg
     1981 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt
     2041 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg
     2101 gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc
     2161 gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt
     2221 gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg
     2281 aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg
     2341 gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg
     2401 ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg
     2461 caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg
     2521 cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt
     2581 ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg
     2641 aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc
     2701 ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc
     2761 cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg
     2821 gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat
     2881 gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat
     2941 tttggcaaag aattccgcca ccggggatcc accggagctt accatggtct ccaaaggaga
     3001 agaagataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc
     3061 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     3121 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct
     3181 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga
     3241 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     3301 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa
     3361 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat
     3421 gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat
     3481 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta
     3541 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat
     3601 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca
     3661 ctccaccggc ggaatggatg aactctataa atgaggtact cctgtgcctt ctagttgcca
     3721 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3781 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3841 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3901 tgctggggat gcggtgggct ctatggaaat aacttcgtat agtgtaggat atacgaagtt
     3961 atataggtat gtcgggaacc tctccgggtc cggggtgagc aagggcgagg agctgttcac
     4021 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     4081 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     4141 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     4201 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     4261 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     4321 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     4381 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     4441 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     4501 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     4561 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtcca agctgagcaa
     4621 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     4681 cactctcggc atggacgagc tgtacaagta agagggcctc tccacttata ctctacactc
     4741 attcttaact tttcgcaaac gtttcacacg gactaatctt tctgaactgt aaatatttaa
     4801 agaaaaaaaa agtagttttg tatcaaccta aatgagtcaa agttcaaaaa tattcatttc
     4861 aatcttttca taattgttga cctaagaata taatacattt gctagtgaca tgtacatact
     4921 gtatatagcc ggattaacgg ttataaagtt ttgtaccatt tattttatga cattttacaa
     4981 tgtaagtttt gaaactaact gttggtagga gaaagtttct tatggaacga ataccctgct
     5041 caacatttaa tcaatctttg tgcctcaaca tactgttgat gtctaagtat gcctcagtgg
     5101 gttgagaaaa tccccattga agatgtcctg tccacctaaa agagaatgat gctgtgcata
     5161 tcacttgata tgtgcaccaa tacctactga atcagaaatg taaggcattg gtgatgtttg
     5221 catttaccct cctgtaagca acactttaac gtcttacatt ttctctgatg atgtcacaca
     5281 aaattatcat gacaaatatt accagagcaa agtgtaacgg ccaacacttt gttcgctcat
     5341 tttacgctgt ctctgacata aggagtgcct gaatagcttg gaaaagtaac atctcctggc
     5401 catcccttca tttaaccaag ctattcaagt attcctatgc cagagcagtg ccaactcttg
     5461 gaggtcccag agtgcagcca atgcctttgt gtggtagttc taaattttaa ttgcacctga
     5521 aaaacctggg cacctaagca atgagccaca gcaaaaagta aagaacaaca acaaaataaa
     5581 gctgttgtta aattttaaac aatattacta attgcccaaa atgtcaattt gatgtagttc
     5641 ttttcatgca agtataaatt caattgttag ttataattgt tggacctcct tgagatagta
     5701 acaacarmwr tyycmwkkgy gmrmykrkyw tssgctcaca attccacaca acatacgagc
     5761 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5821 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5881 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5941 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     6001 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     6061 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6121 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6181 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6241 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6301 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6361 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6421 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6481 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6541 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6601 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6661 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6721 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6781 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6841 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6901 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6961 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     7021 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     7081 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     7141 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     7201 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7261 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7321 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7381 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7441 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7501 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7561 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7621 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7681 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7741 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7801 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtska
     7861 w
//
