LOCUS       sequence-108798-        7918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for gene editing at human VIM locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7918)
  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 7918)
  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-mEmerald-VIM
FEATURES             Location/Qualifiers
     source          1..7918
                     /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             join(2696..2698,2699..2701,2702..3412)
                     /codon_start=1
                     /product="brightly fluorescent monomeric variant of GFP"
                     /label=mEmerald
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2741..2762)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3002..3021)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3349..3370
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4316..4336)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4316..4334)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4355..4371)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4355..4371)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4368..4390)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4379..4395
                     /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(4403..4409,4410..4427,4428..4433))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4586..4603)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4757..5345)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4837..4856)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5516..6307,6308..6376))
                     /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     6139..6158
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6377..6481)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6508..7850
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(7471..7518)
                     /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     7891..7909
                     /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 ttaggtccct cgacagaacc
     1921 tcccctcccc ccaacatctc tccgccaagg caagtcgatg gacagaggcg cgggccggag
     1981 cagcccccct ttccaagcgg gcggcgcgcg aggctgcggc gaggcctgag ccctgcgttc
     2041 ctgcgctgtg cgcgccccca ccccgcgttc caatctcagg cgctctttgt ttctttctcc
     2101 gcgacttcag atctgaggga ttccttactc tttcctcttc ccgctccttt gcccgcgggt
     2161 ctccccgcct gaccgcagcc ccgagaccgc cgcgcacctc ctcccacgcc cctttggcgt
     2221 ggtgccaccg gacccctctg gttcagtccc aggcggaccc ccccctcacc gcgcgacccc
     2281 gcctttttca gcaccccagg gtgagcccag ctcagactat catccggaaa gcccccaaaa
     2341 gtcccagccc agcgctgaag taacgggacc atgcccagtc ccaggccccg gagcaggaag
     2401 gctcgagggc gcccccaccc cacccgccca ccctccccgc ttctcgctag gtccctattg
     2461 gctggcgcgc tccgcggctg ggatggcagt gggaggggac cctctttcct aacggggtta
     2521 taaaaacagc gccctcggcg gggtccagtc ctctgccact ctcgctccga ggtccccgcg
     2581 ccagagacgc agccgcgctc ccaccaccca cacccaccgc gccctcgttc gcctcttctc
     2641 cgggagccag tccgcgccac cgccgccgcc caggccatcg ccaccctccg cagccatggt
     2701 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     2761 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     2821 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     2881 gaccaccttg acctacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca
     2941 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     3001 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     3061 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     3121 ggagtacaac tacaacagcc acaaggtcta tatcaccgcc gacaagcaga agaacggcat
     3181 caaggtgaac ttcaagaccc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     3241 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     3301 gagcacccag tccaagctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     3361 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agggaggttc
     3421 aggaggcagc atgtccacca ggtccgtgag cagcagttcc taccgcagga tgttcggcgg
     3481 cccgggcacc gcgagccgcc cctcctccag ccggagctac gtgactacgt ccacccgcac
     3541 ctacagcctg ggcagcgcgc tgcgccccag caccagccgc agcctctacg cctcgtcccc
     3601 gggcggcgtg tatgccacgc gctcctctgc cgtgcgcctg cggagcagcg tgcccggggt
     3661 gcggctcctg caggactcgg tggacttctc gctggccgac gccatcaaca ccgagttcaa
     3721 gaacacccgc accaacgaga aggtggagct gcaggagctg aatgaccgct tcgccaacta
     3781 catcgacaag gtgcgcttcc tggagcagca gaataagatc ctgctggccg agctcgagca
     3841 gctcaagggc caaggcaagt cgcgcctggg ggacctctac gaggaggaga tgcgggagct
     3901 gcgccggcag gtggaccagc taaccaacga caaagcccgc gtcgaggtgg agcgcgacaa
     3961 cctggccgag gacatcatgc gcctccggga gaagtaaggc tgcgcccatg caagtagctg
     4021 ggcctcggga gggggctgga gggagagggg aacgcccccc cggcccccgc gagagctgcc
     4081 acgcccttgg ggatgtggcc ggggggaggc ctgccaggga gacagcggag agcggggctg
     4141 tggctgtggt ggcgcagccc cgcccagaac ccagaccttg cagttcgcat ttcctcctct
     4201 gtccccacac attgcccaag gacgctccgt ttcaagttac agatttctta aaactaccac
     4261 tttgtgtgca gttgaaggcg cggccgccac cgcggtggag ctccagcttt tgttcccttt
     4321 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4381 gttatccgct cacaattcca cacaacatag gagccggaag cataaagtgt aaagcctggg
     4441 gtgcctaatg agtgaggtaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4501 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4561 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4621 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4681 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4741 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4801 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4861 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4921 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4981 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5041 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5101 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5161 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     5221 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5281 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5341 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5401 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5461 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5521 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5581 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5641 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5701 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5761 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5821 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5881 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5941 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6001 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6061 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6121 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6181 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6241 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6301 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6361 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6421 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6481 gcacatttcc ccgaaaagtg ccacctgaac gaagcatctg tgcttcattt tgtagaacaa
     6541 aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa
     6601 cagaaatgca acgcgaaagc gctattttac caacgaagaa tctgtgcttc atttttgtaa
     6661 aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta
     6721 cagaacagaa atgcaacgcg agagcgctat tttaccaaca aagaatctat acttcttttt
     6781 tgttctacaa aaatgcatcc cgagagcgct atttttctaa caaagcatct tagattactt
     6841 tttttctcct ttgtgcgctc tataatgcag tctcttgata actttttgca ctgtaggtcc
     6901 gttaaggtta gaagaaggct actttggtgt ctattttctc ttccataaaa aaagcctgac
     6961 tccacttccc gcgtttactg attactagcg aagctgcggg tgcatttttt caagataaag
     7021 gcatccccga ttatattcta taccgatgtg gattgcgcat actttgtgaa cagaaagtga
     7081 tagcgttgat gattcttcat tggtcagaaa attatgaacg gtttcttcta ttttgtctct
     7141 atatactacg tataggaaat gtttacattt tcgtattgtt ttcgattcac tctatgaata
     7201 gttcttacta caattttttt gtctaaagag taatactaga gataaacata aaaaatgtag
     7261 aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga tgggtaggtt atatagggat
     7321 atagcacaga gatatatagc aaagagatac ttttgagcaa tgtttgtgga agcggtattc
     7381 gcaatatttt agtagctcgt tacagtccgg tgcgtttttg gttttttgaa agtgcgtctt
     7441 cagagcgctt ttggttttca aaagcgctct gaagttccta tactttctag agaataggaa
     7501 cttcggaata ggaacttcaa agcgtttccg aaaacgagcg cttccgaaaa tgcaacgcga
     7561 gctgcgcaca tacagctcac tgttcacgtc gcacctatat ctgcgtgttg cctgtatata
     7621 tatatacatg agaagaacgg catagtgcgt gtttatgctt aaatgcgtac ttatatgcgt
     7681 ctatttatgt aggatgaaag gtagtctagt acctcctgtg atattatccc attccatgcg
     7741 gggtatcgta tgcttccttc agcactaccc tttagctgtt ctatatgctg ccactcctca
     7801 attggattag tctcatcctt caatgctatc atttcctttg atattggatc atattaagaa
     7861 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
