LOCUS       sequence-173979-        5409 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology arms and linker-mEGFP sequence for C-terminus tagging of 
            human DSP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5409)
  AUTHORS   Roberts B, Haupt A, Tucker A, Grancharova T, Arakaki J, Fuqua MA, 
            Nelson A, Hookway C, Ludmann SA, Mueller IA, Yang R, Horwitz R, 
            Rafelski SM, Gunawardane RN
  TITLE     Systematic gene tagging using CRISPR/Cas9 in human stem cells to 
            illuminate cell organization.
  JOURNAL   Mol Biol Cell. 2017 Oct 15;28(21):2854-2874. doi: 
            10.1091/mbc.E17-03-0209. Epub 2017 Aug 16.
  PUBMED    28814507
REFERENCE   2  (bases 1 to 5409)
  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: 
            "10.1091/mbc.E17-03-0209"; journalName: "Mol Biol Cell"; date: 
            "2017-10-15- 15"; volume: "28"; issue: "21"; pages: "2854-2874"
COMMENT     Sequence Label: AICSDP-9:DSP-mEGFP
FEATURES             Location/Qualifiers
     source          1..5409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(1437..1439,1440..2153)
                     /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(1479..1500)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1740..1759)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2087..2108
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3188..3204)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3188..3204)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3201..3223)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3212..3228
                     /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(3236..3242,3243..3260,3261..3266))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3281..3302
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3419..3436)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3590..4178)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3670..3689)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4349..5140,5141..5209))
                     /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     4972..4991
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5210..5314)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5382..5400
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggattgttg
      421 acaggaagtt ctttgatcag taccgatccg gcagcctcag cctcactcaa tttgctgaca
      481 tgatctcctt gaaaaatggt gtcggcacca gcagcagcat gggcagtggt gtcagcgatg
      541 atgtttttag cagctcccga catgaatcag taagtaagat ttccaccata tccagcgtca
      601 ggaatttaac cataaggagc agctcttttt cagacaccct ggaagaatcg agccccattg
      661 cagccatctt tgacacagaa aacctggaga aaatctccat tacagaaggt atagagcggg
      721 gcatcgttga cagcatcacg ggtcagaggc ttctggaggc tcaggcctgc acaggtggca
      781 tcatccaccc aaccacgggc cagaagctgt cacttcagga cgcagtctcc cagggtgtga
      841 ttgaccaaga catggccacc aggctgaagc ctgctcagaa agccttcata ggcttcgagg
      901 gtgtgaaggg aaagaagaag atgtcagcag cagaggcagt gaaagaaaaa tggctcccgt
      961 atgaggctgg ccagcgcttc ctggagttcc agtacctcac gggaggtctt gttgacccgg
     1021 aagtgcatgg gaggataagc accgaagaag ccatccggaa ggggttcata gatggccgcg
     1081 ccgcacagag gctgcaagac accagcagct atgccaaaat cctgacctgc cccaaaacca
     1141 aattaaaaat atcctataag gatgccataa atcgctccat ggtagaagat atcactgggc
     1201 tgcgccttct ggaagccgcc tccgtgtcgt ccaagggctt acccagccct tacaacatgt
     1261 cttcggctcc gggctcccgc tccggctccc gctcgggatc tcgctccgga tctcgctccg
     1321 ggtcccgcag tgggtcccgg agaggaagct ttgacgccac agggaattct tcctactctt
     1381 attcatactc atttagcagt agtagcattg ggcaccacga cccccccgtt gctacggtga
     1441 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1501 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     1561 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1621 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     1681 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     1741 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     1801 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1861 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     1921 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     1981 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2041 gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2101 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taatagtagt
     2161 cagttgcgag tggttgctat accttgactt catttatatg aatttccact ttattaaata
     2221 atagaaaaga aaatcccggt gcttgcagta gagtgatagg acattctatg cttacagaaa
     2281 atatagccat gattgaaatc aaatagtaaa ggctgttctg gctttttatc ttcttagctc
     2341 atcttaaata agcagtacac ttggatgcag tgcgtctgaa gtgctaatca gttgtaacaa
     2401 tagcacaaat cgaacttagg atttgtttct tctcttctgt gtttcgattt ttgatcaatt
     2461 ctttaatttt ggaagcctat aatacagttt tctattcttg gagataaaaa ttaaatggat
     2521 cactgatatt ttagtcattc tgcttctcat ctaaatattt ccatattctg tattaggaga
     2581 aaattaccct cccagcacca gcccccctct caaaccccca acccaaaacc aagcattttg
     2641 gaatgagtct cctttagttt cagagtgtgg attgtataac ccatatactc ttcgatgtac
     2701 ttgtttggtt tggtattaat ttgactgtgc atgacagcgg caatcttttc tttggtcaaa
     2761 gttttctgtt tattttgctt gtcatattcg atgtacttta aggtgtcttt atgaagtttg
     2821 ctattctggc aataaacttt tagacttttg aagtgtttgt gttttaattt aatatgttta
     2881 taagcatgta taaacattta gcatattttt atcataggtc taaaaatatt tgtttactaa
     2941 atacctgtga agaaatacca ttaaaaaact atttggttct gaattcttac tagaaggtgg
     3001 tcttttgaag ttagtccttt cggtacttct cagatgcctg tcatgtaccc gatggagtcc
     3061 ttggaaagaa ggcctgtgta aagaggccag cctggaggtc aataacctgt tctagtttat
     3121 tctggacatt gagtaccaag tagcattggc aaaatcccaa tggcgcgccg agcttggcgt
     3181 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     3241 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     3301 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3361 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3421 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3481 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3541 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3601 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3661 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3721 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3781 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3841 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3901 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3961 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4021 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4081 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4141 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4201 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4261 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4321 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4381 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4441 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4501 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4561 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4621 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4681 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4741 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4801 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4861 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4921 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4981 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5041 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5101 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5161 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5221 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5281 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5341 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     5401 cctttcgtc
//
