LOCUS       sequence-441601-        5455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology arms and linker-mEGFP sequence for N-terminus (second exon)
            tagging of human TUBA1B.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5455)
  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 5455)
  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-4:TUBA1B-mEGFP
FEATURES             Location/Qualifiers
     source          1..5455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(2074..2076,2077..2787)
                     /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(2116..2137)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2377..2396)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2724..2745
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3830..3847)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3830..3846)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3839..3861)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4055..4074)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4174..4196
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4234..4252)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4320..4424
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4425..4493,4494..5285)
                     /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     complement(4643..4662)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg catgcctgca
     1021 ggtcgactct agagtgcatg cctgcaggtc gactgagtgt tctttttttg atgaaagcaa
     1081 taagaggact gcggaagagc tccctgtcaa tgtaccgctc tacaccagtg tattacgaca
     1141 gttcgtacac aacagtctgt agaggccacc tgtctctccc tgctgcgtta ggaattcagg
     1201 ggagcaggtg gtggcagtaa gggattttga gggaacggaa atcggatctt gacccagatc
     1261 tgggccgccg ataatctcct actgcgctca gactgctgtg gaggtgttag gctgagcccg
     1321 atgccggcag gcaagggagg atgggcggct tgggcagcgc ctttgcagac gtggccattt
     1381 cgtgcctctg cagcaccgcc ggggggcgca agagcgcgcg cccggaattg ctcattcatc
     1441 ctgtgccgca gagccccgcc ccttgtccct gcggacagac atttcttctg cgctggtctg
     1501 gccacgtgct tcctgtgcta ggagctgccc ggaaatgtga ccacctagtc taaagtgggc
     1561 ttctggggcc tgagcgctgg atggatgccc accttcctgt cttggtcctc caaaggagga
     1621 agctgtgact gagctgtctt ggtctggaag gaggccttcc cggtttagga tgggaaggta
     1681 acattcatta aaagcaacgt agactatagt gtagctgttc tcaaaagtag tacatcttag
     1741 aaaaggatct ttagaaaaga tcgctttaga aaaggaaatt cgttttcaga ttacgtgagt
     1801 agcctaggta acacagccag acctcatctc cacaaaaaaa atgaaaaaat tagccagctt
     1861 ggtggtctgt gcctgtggtc ccagctgctc cagaggctga ggtgggggga tgactggagc
     1921 ctaggctgca gtgagcctag atggcatcac tgcactcaag actgggcgac agaccttatc
     1981 tctaaaaaaa taaagattgc atgagtattt tgttccactt gacagtcatc aatagattgg
     2041 tttaaattgt gatatctttt ttacttaccg caggtgagca agggcgagga gctgttcacc
     2101 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2161 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     2221 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     2281 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     2341 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     2401 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2461 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     2521 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     2581 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     2641 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccaa gctgagcaaa
     2701 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     2761 actctcggca tggacgagct gtacaaggga ggttcaggag gcagcgagtg catctccatc
     2821 cacgttggcc aggctggtgt ccagattggc aatgcctgct gggagctcta ctgcctggaa
     2881 cacggcatcc agcccgatgg ccagatgcca agtgacaaga ccattggggg aggagatgac
     2941 tccttcaaca ccttcttcag tgagacgggc gctggcaagc acgtgccccg ggctgtgttt
     3001 gtagacttgg aacccacagt cattggtgag ttgacctcag taacctgaga tcccaggatg
     3061 ctgggacagg aggtctgtcc aggggcttct cttgtcactc actcactccc tccgtccttc
     3121 tctccctcct ccagatgaag ttcgcactgg cacctaccgc cagctcttcc accctgagca
     3181 gctcatcaca ggcaaggaag atgctgccaa taactatgcc cgagggcact acaccattgg
     3241 caaggagatc attgaccttg tgttggaccg aattcgcaag ctggtaagca ccacatataa
     3301 atatgcattt aatgtggtgt gatagttcca gtgcaagttg ggtggagtga ctgacatcat
     3361 tcattctttg gcacctacca aaatgtggaa taggctgctt gctatattaa ttggacttct
     3421 aaatcagata gtccctaggt tatggacagt ttgtggatat gtctgttttg ccaattcctt
     3481 gtgcttacat cagtgagata tggttcgtaa tctaaaaagt tgaaatagaa attctaagat
     3541 aatgtgtcct ggcattaaaa tattacattt ttttattccc ctacaggctg accagtgcac
     3601 cggtcttcag ggcttcttgg ttttccacag ctttggtggg ggaactggtt ctgggttcac
     3661 ctccctgctc atggaacgtc tctcagttga ttatggcaag aagtccaagc tggagttctc
     3721 catttaccca gcaccccagg tttccacagc tgtagttgag ccctacaact ccatcctcac
     3781 cacccacacc accctggagc actcgtaccg agctcgaatt cactaattca ctggccgtcg
     3841 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     3901 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     3961 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     4021 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4081 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4141 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4201 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     4261 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     4321 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4381 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4441 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4501 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4561 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4621 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     4681 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     4741 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4801 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     4861 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4921 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     4981 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5041 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5101 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5161 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5221 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5281 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5341 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5401 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
