LOCUS       sequence-405875-        6373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet-inducible expression of shRNA targeting gamma-tubulin in human 
            cells, retroviral.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6373)
  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 6373)
  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: pSuperior.retro.puro-sh-gamma-tubulin
FEATURES             Location/Qualifiers
     source          1..6373
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     1421..1437
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1422..1438
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1504..1522
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(1568..1587)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     promoter        1733..2232
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1768..1791)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2165..2184
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2257..2856
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2257..2276)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2753..2773
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    3442..3458
                     /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(3466..3472,3473..3490,3491..3496))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3511..3532
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3649..3666)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3820..4408)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3900..3919)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4579..5370,5371..5439))
                     /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     5202..5221
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5440..5544)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5612..5630
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5668..5690)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5790..5809
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6003..6025
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6153..6172
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(6372..6373,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatcgatctc tcgaggtcga cggtatcgat
     1441 aagcttaaaa aaggaggaca tgttcaagga tctcttgaat ccttgaacat gtcctcctgg
     1501 ggatctctat cactgatagg gaacttataa gattcccaaa tccaaagaca tttcacgttt
     1561 atggtgattt cccagaacac atagcgacat gcaaatattg cagggcgcca ctcccctgtc
     1621 cctcacagcc atcttcctgc cagggcgcac gcgcgctggg tgttcccgcc tagtgacact
     1681 gggcccgcga ttccttggag cgggttgatg acgtcagcgt tcgaattcta ccgggtaggg
     1741 gaggcgcttt tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg
     1801 cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg
     1861 gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc
     1921 ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact
     1981 agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc
     2041 ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc
     2101 gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc
     2161 ccggcattct gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct
     2221 ccgggccttt cgacctgcag cccaagctag cttaccatga ccgagtacaa gcccacggtg
     2281 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     2341 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     2401 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     2461 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     2521 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     2581 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     2641 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     2701 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     2761 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     2821 cgcacctggt gcatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc
     2881 ccgaccgaaa ggagcgcacg accccatgca tcgataaaat aaaagatttt atttagtctc
     2941 cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg gcaagctaga gaaccatcag
     3001 atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat
     3061 cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca
     3121 caacccctca ctcggcgcgc cagtcctccg atagactgcg tcgcccgggt acccgtgtat
     3181 ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct
     3241 cctctgagtg attgactacc cgtcagcggg ggtctttcat gggtaacagt ttcttgaagt
     3301 tggagaacaa cattctgagg gtaggagtcg aatattaagt aatcctgact caattagcca
     3361 ctgttttgaa tccacatact ccaatactcc tgaaatagtt cattatggac agcgcagaaa
     3421 gagctgggga gaattgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3481 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3541 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3601 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3661 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3721 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3781 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3841 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3901 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3961 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4021 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     4081 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4141 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4201 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4261 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4321 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4381 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4441 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4501 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4561 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4621 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4681 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4741 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4801 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4861 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4921 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4981 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     5041 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     5101 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     5161 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     5221 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5281 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5341 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5401 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5461 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5521 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     5581 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     5641 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     5701 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     5761 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     5821 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca
     5881 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     5941 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     6001 ttcccagtca cgacgttgta aaacgacggc gcaaggaatg gtgcatgcaa ggagatggcg
     6061 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     6121 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     6181 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gcgattagtc
     6241 caatttgtta aagacaggat atcagtggtc caggctctag ttttgactca acaatatcac
     6301 cagctgaagc ctatagagta cgagccatag ataaaataaa agattttatt tagtctccag
     6361 aaaaaggggg gaa
//
