LOCUS       sequence-108813-        7718 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for gene editing at human TUBA1B locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7718)
  AUTHORS   Gan Z, Ding L, Burckhardt CJ, Lowery J, Zaritsky A, Sitterley K, 
            Mota A, Costigliola N, Starker CG, Voytas DF, Tytell J, Goldman RD, 
            Danuser G
  TITLE     Vimentin Intermediate Filaments Template Microtubule Networks to 
            Enhance Persistence in Cell Polarity and Directed Migration.
  JOURNAL   Cell Syst. 2016 Sep 21. pii: S2405-4712(16)30262-9. doi: 
            10.1016/j.cels.2016.08.007.
  PUBMED    27667364
REFERENCE   2  (bases 1 to 7718)
  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"; journalName: "Cell Syst. 
            2016 Sep 21. pii: S2405-4712(16)30262-9. doi: 
            10.1016/j.cels.2016.08.007."
COMMENT     Sequence Label: pRS-mTagRFP-TUBA1B
FEATURES             Location/Qualifiers
     source          1..7718
                     /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"
     promoter        187..467
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             468..1142
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      complement(1241..1696)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1378..1399)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1590..1609
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1826..1848
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1840..1857
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1841..1857
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1867..1886
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1867..1885
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2632..3360
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     primer_bind     complement(4116..4136)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4116..4134)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4155..4171)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4155..4171)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4168..4190)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4179..4195
                     /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(4203..4209,4210..4227,4228..4233))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4386..4403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4557..5145)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4637..4656)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5316..6107,6108..6176))
                     /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     5939..5958
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6177..6281)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6308..7650
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(7271..7318)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     7691..7709
                     /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 accataaacg acattactat atatataata taggaagcat ttaatagaca gcatcgtaat
      241 atatgtgtac tttgcagtta tgacgccaga tggcagtagt ggaagatatt ctttattgaa
      301 aaatagcttg tcaccttacg tacaatcttg atccggagct tttctttttt tgccgattaa
      361 gaattaattc ggtcgaaaaa agaaaaggag agggccaaga gggagggcat tggtgactat
      421 tgagcacgtg agtatacgtg attaagcaca caaaggcagc ttggagtatg tctgttatta
      481 atttcacagg tagttctggt ccattggtga aagtttgcgg cttgcagagc acagaggccg
      541 cagaatgtgc tctagattcc gatgctgact tgctgggtat tatatgtgtg cccaatagaa
      601 agagaacaat tgacccggtt attgcaagga aaatttcaag tcttgtaaaa gcatataaaa
      661 atagttcagg cactccgaaa tacttggttg gcgtgtttcg taatcaacct aaggaggatg
      721 ttttggctct ggtcaatgat tacggcattg atatcgtcca actgcatgga gatgagtcgt
      781 ggcaagaata ccaagagttc ctcggtttgc cagttattaa aagactcgta tttccaaaag
      841 actgcaacat actactcagt gcagcttcac agaaacctca ttcgtttatt cccttgtttg
      901 attcagaagc aggtgggaca ggtgaacttt tggattggaa ctcgatttct gactgggttg
      961 gaaggcaaga gagccccgaa agcttacatt ttatgttagc tggtggactg acgccagaaa
     1021 atgttggtga tgcgcttaga ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg
     1081 agacaaatgg tgtaaaagac tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat
     1141 aggttattac tgagtagtat ttatttaagt attgtttgtg cacttgccta tgcggtgtga
     1201 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt
     1261 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     1321 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     1381 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     1441 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     1501 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     1561 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     1621 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     1681 ccgctacagg gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     1741 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     1801 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag
     1861 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg cggcttgggc agcgcctttg
     1921 cagacgtggc catttcgtgc ctctgcagca ccgccggggg gcgcaagagc gcgcgcccgg
     1981 aattgctcat tcatcctgtg ccgcagagcc ccgccccttg tccctgcgga cagacatttc
     2041 ttctgcgctg gtctggccac gtgcttcctg tgctaggagc tgcccggaaa tgtgaccacc
     2101 tagtctaaag tgggcttctg gggcctgagc gctggatgga tgcccacctt cctgtcttgg
     2161 tcctccaaag gaggaagctg tgactgagct gtcttggtct ggaaggaggc cttcccggtt
     2221 taggatggga aggtaacatt cattaaaagc aacgtagact atagtgtagc tgttctcaaa
     2281 agtagtacat cttagaaaag gatctttaga aaagatcgct ttagaaaagg aaattcgttt
     2341 tcagattacg tgagtagcct aggtaacaca gccagacctc atctccacaa aaaaaatgaa
     2401 aaaattagcc agcttggtgg tctgtgcctg tggtcccagc tgctccagag gctgaggtgg
     2461 ggggatgact ggagcctagg ctgcagtgag cctagatggc atcactgcac tcaagcctgg
     2521 gcgacagacc ttatctctaa aaaaataaag attgcatgag tattttgttc cacttgacag
     2581 tcatcaatag attggtttaa attgtgatat cttttttcct tcccgcagcg tgtgtctaag
     2641 ggcgaagagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtgaac
     2701 aaccaccact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc
     2761 atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctacc
     2821 agcttcatgt acggcagcag aaccttcatc aaccacaccc agggcatccc cgatttcttt
     2881 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
     2941 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag
     3001 atcagagggg tgaacttccc atccaacggc cctgtgatgc agaagaaaac actcggctgg
     3061 gaggccaaca ccgagatgct gtaccccgct gacggcggcc tggaaggcag aaccgacatg
     3121 gccctgaagc tcgtgggcgg gggccacctg atctgcaact tcaagaccac atacagatcc
     3181 aagaaacccg ctaagaacct caagatgccc ggcgtctact atgtggacca cagactggaa
     3241 agaatcaagg aggccgacaa agagacctac gtcgagcagc acgaggtggc tgtggccaga
     3301 tactgcgacc tccctagcaa actggggcac aaacttaatg gcatggacga gctgtacaag
     3361 ggaggttcag gaggcagccg tgagtgcatc tccatccacg ttggccaggc tggtgtccag
     3421 attggcaatg cctgctggga gctctactgc ctggaacacg gcatccagcc cgatggccag
     3481 atgccaagtg acaagaccat tgggggagga gatgactcct tcaacacctt cttcagtgag
     3541 acgggcgctg gcaagcacgt gccccgggct gtgtttgtag acttggaacc cacagtcatt
     3601 ggtgagttga cctcagtaac ctgagatccc aggatgctgg gacaggaggt ctgtccaggg
     3661 gcttctcttg tcactcactc actccctccg tccttctctc cctcctccag atgaagttcg
     3721 cactggcacc taccgccagc tcttccaccc tgagcagctc atcacaggca aggaagatgc
     3781 tgccaataac tatgcccgag ggcactacac cattggcaag gagatcattg accttgtgtt
     3841 ggaccgaatt cgcaagctgg taagcaccac atataaatat gcatttaatg tggtgtgata
     3901 gttccagtgc aagttgggtg gagtgactga catcattcat tctttggcac ctaccaaaat
     3961 gtggaatagg ctgcttgcta tattaattgg acttctaaat cagatagtcc ctaggttatg
     4021 gacagtttgt ggatatgtct gttttgccaa ttccttgtgc ttacatcagt gagatatggg
     4081 cggccgccac cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc
     4141 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4201 cacaacatag gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgaggtaa
     4261 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4321 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4381 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4441 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4501 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4561 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4621 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4681 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4741 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4801 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4861 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4921 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4981 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5041 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5101 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5161 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5221 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5281 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5341 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5401 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5461 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5521 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5581 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5641 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5701 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5761 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5821 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5881 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5941 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6001 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6061 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6121 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6181 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6241 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6301 ccacctgaac gaagcatctg tgcttcattt tgtagaacaa aaatgcaacg cgagagcgct
     6361 aatttttcaa acaaagaatc tgagctgcat ttttacagaa cagaaatgca acgcgaaagc
     6421 gctattttac caacgaagaa tctgtgcttc atttttgtaa aacaaaaatg caacgcgaga
     6481 gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg
     6541 agagcgctat tttaccaaca aagaatctat acttcttttt tgttctacaa aaatgcatcc
     6601 cgagagcgct atttttctaa caaagcatct tagattactt tttttctcct ttgtgcgctc
     6661 tataatgcag tctcttgata actttttgca ctgtaggtcc gttaaggtta gaagaaggct
     6721 actttggtgt ctattttctc ttccataaaa aaagcctgac tccacttccc gcgtttactg
     6781 attactagcg aagctgcggg tgcatttttt caagataaag gcatccccga ttatattcta
     6841 taccgatgtg gattgcgcat actttgtgaa cagaaagtga tagcgttgat gattcttcat
     6901 tggtcagaaa attatgaacg gtttcttcta ttttgtctct atatactacg tataggaaat
     6961 gtttacattt tcgtattgtt ttcgattcac tctatgaata gttcttacta caattttttt
     7021 gtctaaagag taatactaga gataaacata aaaaatgtag aggtcgagtt tagatgcaag
     7081 ttcaaggagc gaaaggtgga tgggtaggtt atatagggat atagcacaga gatatatagc
     7141 aaagagatac ttttgagcaa tgtttgtgga agcggtattc gcaatatttt agtagctcgt
     7201 tacagtccgg tgcgtttttg gttttttgaa agtgcgtctt cagagcgctt ttggttttca
     7261 aaagcgctct gaagttccta tactttctag agaataggaa cttcggaata ggaacttcaa
     7321 agcgtttccg aaaacgagcg cttccgaaaa tgcaacgcga gctgcgcaca tacagctcac
     7381 tgttcacgtc gcacctatat ctgcgtgttg cctgtatata tatatacatg agaagaacgg
     7441 catagtgcgt gtttatgctt aaatgcgtac ttatatgcgt ctatttatgt aggatgaaag
     7501 gtagtctagt acctcctgtg atattatccc attccatgcg gggtatcgta tgcttccttc
     7561 agcactaccc tttagctgtt ctatatgctg ccactcctca attggattag tctcatcctt
     7621 caatgctatc atttcctttg atattggatc atattaagaa accattatta tcatgacatt
     7681 aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
