LOCUS       sequence-174163-        5399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology arms and linker-mEGFP sequence for N-terminus tagging of 
            human ACTB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5399)
  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 5399)
  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-15:ACTB-mEGFP
FEATURES             Location/Qualifiers
     source          1..5399
                     /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(1412..1414,1415..1417,1418..2128)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1457..1478)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1718..1737)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2065..2086
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3178..3194)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3178..3194)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3191..3213)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3202..3218
                     /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(3226..3232,3233..3250,3251..3256))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3271..3292
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3409..3426)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3580..4168)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3660..3679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4339..5130,5131..5199))
                     /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     4962..4981
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5200..5304)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5372..5390
                     /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 ggatgctcga
      421 gcggccgcgg cggcgcccta taaaacccag cggcgcgacg cgccaccacc gccgagaccg
      481 cgtccgcccc gcgagcacag agcctcgcct ttgccgatcc gccgcccgtc cacacccgcc
      541 gccaggtaag cccggccagc cgaccggggc aggcggctca cggcccggcc gcaggcggcc
      601 gcggcccctt cgcccgtgca gagccgccgt ctgggccgca gcggggggcg catggggggg
      661 gaaccggacc gccgtggggg gcgcgggaga agcccctggg cctccggaga tgggggacac
      721 cccacgccag ttcggaggcg cgaggccgcg ctcgggaggc gcgctccggg ggtgccgctc
      781 tcggggcggg ggcaaccggc ggggtctttg tctgagccgg gctcttgcca atggggatcg
      841 cagggtgggc gcggcggagc ccccgccagg cccggtgggg gctggggcgc cattgcgcgt
      901 gcgcgctggt cctttgggcg ctaactgcgt gcgcgctggg aattggcgct aattgcgcgt
      961 gcgcgctggg actcaaggcg ctaactgcgc gtgcgttctg gggcccgggg tgccgcggcc
     1021 tgggctgggg cgaaggcggg ctcggccgga aggggtgggg tcgccgcggc tcccgggcgc
     1081 ttgcgcgcac ttcctgcccg agccgctggc cgcccgaggg tgtggccgct gcgtgcgcgc
     1141 gcgccgaccc ggcgctgttt gaaccgggcg gaggcggggc tggcgcccgg ttgggagggg
     1201 gttggggcct ggcttcctgc cgcgcgccgc ggggacgcct ccgaccagtg tttgcctttt
     1261 atggtaataa cgcggccggc ccggcttcct ttgtccccaa tctgggcgcg cgccggcgcc
     1321 ccctggcggc ctaaggactc ggcgcgccgg aagtggccag ggcgggggcg acctcggctc
     1381 acagcgcgcc cggctattct cgcaactgac aatggtgagc aagggcgagg agctgttcac
     1441 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1501 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1561 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1621 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1681 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1741 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1801 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1861 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1921 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1981 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtcca agctgagcaa
     2041 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2101 cactctcggc atggacgagc tgtacaaggc cggctccggt accgatgatg atatcgcagc
     2161 gctcgtcgtc gacaacggct ccggcatgtg caaggccggc ttcgcgggcg acgatgcccc
     2221 ccgggccgtc ttcccctcca tcgtggggcg ccccaggcac caggtagggg agctggctgg
     2281 gtggggcagc cccgggagcg ggcgggaggc aagggcgctt tctctgcaca ggagcctccc
     2341 ggtttccggg gtgggggctg cgcccgtgct cagggcttct tgtcctttcc ttcccagggc
     2401 gtgatggtgg gcatgggtca gaaggattcc tatgtgggcg acgaggccca gagcaagaga
     2461 ggcatcctca ccctgaagta ccccatcgag cacggcatcg tcaccaactg ggacgacatg
     2521 gagaaaatct ggcaccacac cttctacaat gagctgcgtg tggctcccga ggagcacccc
     2581 gtgctgctga ccgaggcccc cctgaacccc aaggccaacc gcgagaagat gacccaggtg
     2641 agtggcccgc tacctcttct ggtggccgcc tccctccttc ctggcctccc ggagctgcgc
     2701 cctttctcac tggttctctc ttctgccgtt ttccgtagga ctctcttctc tgacctgagt
     2761 ctcctttgga actctgcagg ttctatttgc tttttcccag atgagctctt tttctggtgt
     2821 ttgtctctct gactaggtgt ctaagacagt gttgtgggtg taggtactaa cactggctcg
     2881 tgtgacaagg ccatgaggct ggtgtaaagc ggccttggag tgtgtattaa gtaggcgcac
     2941 agtaggtctg aacagactcc ccatcccaag accccagcac acttagccgt gttctttgca
     3001 ctttctgcat gtcccccgtc tggcctggct gtccccagtg gcttccccag tgtgacatgg
     3061 tgtatctctg ccttacagat catgtttgag accttcaaca ccccagccat gtacgttgct
     3121 atccaggctg tgctatccct gtaatcccaa tggcgcgccg agcttggcgt aatcatggtc
     3181 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3241 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3301 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3361 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3421 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3481 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3541 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3601 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3661 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3721 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3781 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3841 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3901 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3961 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4021 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4081 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4141 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4201 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4261 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4321 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4381 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4441 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4501 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4561 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4621 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4681 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4741 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4801 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4861 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4921 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4981 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5041 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5101 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5161 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5221 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5281 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     5341 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
