LOCUS       sequence-405874-        7166 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet-inducible expression of WT gamma-tubulin in human cells, harbors
            silent mutations in shRNA target site, FLP-In mediated insertion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7166)
  AUTHORS   Berman AY, Wieczorek M, Aher A, Olinares PDB, Chait BT, Kapoor TM
  TITLE     A nucleotide binding-independent role for gamma-tubulin in 
            microtubule capping and cell division.
  JOURNAL   J Cell Biol. 2023 Mar 6;222(3):e202204102. doi: 
            10.1083/jcb.202204102. Epub 2023 Jan 25.
  PUBMED    36695784
REFERENCE   2  (bases 1 to 7166)
  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: "J Cell 
            Biol."; date: "2023-03-6"; volume: "222(3):e202204102. doi"; pages: 
            " 10.1083/jcb.202204102. Epub 2023 Jan 25"
COMMENT     Sequence Label: pCDNA5/FRT/TO–gamma-tubulinWT-RNAi-resistant-GFP
FEATURES             Location/Qualifiers
     source          1..7166
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(2367..2369,2370..2372,2373..3083)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2367..2369,2370..2372,2373..3083)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2412..2433)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2673..2692)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3020..3041
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3118..3135)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3124..3348
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3481..3500)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3632..3679
                     /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)."
     CDS             3687..4709
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3687..4709
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4839..4960
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4876..4895)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4930..4949
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5009..5025)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5009..5025)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5022..5044)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5033..5049
                     /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(5057..5063,5064..5081,5082..5087))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5102..5123
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5240..5257)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5411..5999)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5491..5510)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6170..6961,6962..7030))
                     /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     6793..6812
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7031..7135)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg gtggcatgcc gagggaaatc atcaccctac
     1021 agttgggcca gtgcggcaat cagattgggt tcgagttctg gaaacagctg tgcgccgagc
     1081 atggtatcag ccccgagggc atcgtggagg agttcgccac cgagggcact gaccgcaagg
     1141 acgtcttttt ctaccaggca gacgatgagc actacatccc ccgggccgtg ctgctggact
     1201 tggaaccccg ggtgatccac tccatcctca actcccccta tgccaagctc tacaacccag
     1261 agaacatcta cctgtcggaa catggaggag gagctggcaa caactgggcc agcggattct
     1321 cccagggaga aaagatccat gaggacattt ttgacatcat agaccgggag gcagatggta
     1381 gtgacagtct agagggcttt gtgctgtgtc actccattgc tggggggaca ggctctggac
     1441 tgggttccta cctcttagaa cggctgaatg acaggtatcc taagaagctg gtgcagacat
     1501 actcagtgtt tcccaaccag gacgagatga gcgatgtggt ggtccagcct tacaattcac
     1561 tcctcacact caagaggctg acgcagaatg cagactgtgt ggtggtgctg gacaacacag
     1621 ccctgaaccg gattgccaca gaccgcctgc acatccagaa cccatccttc tcccagatca
     1681 accagctggt gtctaccatc atgtcagcca gcaccaccac cctgcgctac cctggctaca
     1741 tgaacaatga cctcatcggc ctcatcgcct cgctcattcc caccccacgg ctccacttcc
     1801 tcatgaccgg ctacacccct ctcactacgg accagtcagt ggccagcgtg aggaagacta
     1861 cggtcctgga tgtcatgagg cggctgctgc agcccaagaa cgtgatggtg tccacaggcc
     1921 gagaccgcca gaccaaccac tgctacatcg ccatcctcaa catcatccag ggagaggtgg
     1981 accccaccca ggtccacaag agcttgcaga ggatccggga acgcaagttg gccaacttca
     2041 tcccgtgggg ccccgccagc atccaggtgg ccctgtcgag gaagtctccc tacctgccct
     2101 cggcccaccg ggtcagcggg ctcatgatgg ccaaccacac cagcatctcc tcgctcttcg
     2161 agagaacctg tcgccagtat gacaagctgc gtaagcggga ggccttcctg gagcagttcc
     2221 gcaaggaaga tatgtttaaa gacaactttg atgagatgga cacatccagg gagattgtgc
     2281 agcagctcat cgatgagtac catgcggcca cacggccaga ctacatctcc tggggcaccc
     2341 aggagcaggg agggtctggc ggccgcatgg tgagcaaggg cgaggagctg ttcaccgggg
     2401 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2461 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2521 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     2581 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2641 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2701 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2761 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     2821 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2881 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2941 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     3001 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3061 tcggcatgga cgagctgtac aagaagcttt aagggcccgt ttaaacccgc tgatcagcct
     3121 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3181 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3241 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3301 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     3361 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3421 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3481 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3541 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3601 aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc
     3661 tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga
     3721 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
     3781 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
     3841 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
     3901 cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc
     3961 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
     4021 agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
     4081 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
     4141 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
     4201 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
     4261 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
     4321 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
     4381 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
     4441 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
     4501 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
     4561 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
     4621 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
     4681 ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg
     4741 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4801 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4861 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4921 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     4981 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5041 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5101 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5161 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5221 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5281 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5341 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5401 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5461 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5521 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5581 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5641 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5701 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5761 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5821 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5881 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5941 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6001 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6061 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6121 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6181 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6241 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6301 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6361 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6421 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6481 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6541 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6601 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6661 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6721 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6781 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6841 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6901 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6961 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7021 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7081 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7141 ttccccgaaa agtgccacct gacgtc
//
