LOCUS       sequence-363922-        7625 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral empty vector for doxycycline-inducible expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7625)
  AUTHORS   Gugnoni M, Manzotti G, Vitale E, Sauta E, Torricelli F, Reggiani F, 
            Pistoni M, Piana S, Ciarrocchi A
  TITLE     OVOL2 impairs RHO GTPase signaling to restrain mitosis and 
            aggressiveness of Anaplastic Thyroid Cancer.
  JOURNAL   J Exp Clin Cancer Res. 2022 Mar 25;41(1):108. doi: 
            10.1186/s13046-022-02316-2.
  PUBMED    35337349
REFERENCE   2  (bases 1 to 7625)
  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 Exp Clin 
            Cancer Res."; date: "2022-03-25"; volume: "41(1):108. doi"; pages: "
            10.1186/s13046-022-02316-2"
COMMENT     Sequence Label: pCW empty vector
FEATURES             Location/Qualifiers
     source          1..7625
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    41..158
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        212..526
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    220..238
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    256..274
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    291..309
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    327..345
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    363..381
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    398..416
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    434..452
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     499..523
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        567..1077
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     996..1014
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1087..1683
                     /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(1087..1106)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1583..1603
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1684..1737
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1738..2355,2356..2361,2362..2397,2398..2400,2401..2436,
                     2437..2439,2440..2475,2476..2484)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(2524..2544)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2942..2953)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3158..3391
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3463..3584
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3500..3519)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3554..3573
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3624..3759
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3759..3830
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3831..3899,3900..4691)
                     /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(4049..4068)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4862..5450
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5351..5370
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5604..5621
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5738..5759
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5774..5779,5780..5797,5798..5804)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5812..5828
                     /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     5817..5839
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5836..5852
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5836..5852
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5871..5891
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5873..5891
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5919..6145
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6146..6326
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6373..6498
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6991..7224
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7409..7453
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
ORIGIN
        1 aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
       61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
      121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
      181 gagatccact ttggccgcga atcgatatgt cgagtttact ccctatcagt gatagagaac
      241 gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag
      301 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt
      361 actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt
      421 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg
      481 ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gaattggcta
      541 gcgaattcgt taacacgcgt ggatccgggg ttggggttgc gccttttcca aggcagccct
      601 gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac
      661 cctgggtctc gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc
      721 cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg
      781 ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac
      841 ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc
      901 tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt
      961 ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg
     1021 gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagcaa
     1081 ttcaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     1141 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     1201 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     1261 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     1321 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     1381 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     1441 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt
     1501 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     1561 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     1621 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     1681 gccgaaggta gaggttctct cctcacttgt ggtgatgttg aagaaaaccc tggtccaatg
     1741 tctagactgg acaagagcaa agtcataaac ggagctctgg aattactcaa tggtgtcggt
     1801 atcgaaggcc tgacgacaag gaaactcgct caaaagctgg gagttgagca gcctaccctg
     1861 tactggcacg tgaagaacaa gcgggccctg ctcgatgccc tgccaatcga gatgctggac
     1921 aggcatcata cccacttctg ccccctggaa ggcgagtcat ggcaagactt tctgcggaac
     1981 aacgccaagt cataccgctg tgctctcctc tcacatcgcg acggggctaa agtgcatctc
     2041 ggcacccgcc caacagagaa acagtacgaa accctggaaa atcagctcgc gttcctgtgt
     2101 cagcaaggct tctccctgga gaacgcactg tacgctctgt ccgccgtggg ccactttaca
     2161 ctgggctgcg tattggagga acaggagcat caagtagcaa aagaggaaag agagacacct
     2221 accaccgatt ctatgccccc acttctgaga caagcaattg agctgttcga ccggcaggga
     2281 gccgaacctg ccttcctttt cggcctggaa ctaatcatat gtggcctgga gaaacagcta
     2341 aagtgcgaaa gcggcgggcc gaccgacgcc cttgacgatt ttgacttaga catgctccca
     2401 gccgatgccc ttgacgactt tgaccttgat atgctgcctg ctgacgctct tgacgatttt
     2461 gaccttgaca tgctccccgg gtaactaagt aaggatcgat ccaagatatc gtattcttaa
     2521 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     2581 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     2641 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     2701 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     2761 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     2821 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc
     2881 atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     2941 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     3001 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc
     3061 tgtttcgcct cggcgtccgg actagaggta cctttaagac caatgactta caaggcagct
     3121 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     3181 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     3241 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     3301 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     3361 cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag
     3421 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
     3481 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     3541 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
     3601 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3661 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     3721 agctattcca gaagtagtga ggaggctttt ttggaggcct ttcaaatatg tatccgctca
     3781 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3841 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3901 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3961 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4021 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4081 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4141 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4201 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4261 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4321 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     4381 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4441 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4501 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     4561 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     4621 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4681 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4741 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4801 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4861 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4921 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4981 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5041 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5101 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5161 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5221 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5281 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5341 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5401 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5461 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5521 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5581 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     5641 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     5701 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     5761 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     5821 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
     5881 ctcactaaag ggaacaaaag ctggagctgc aagcttaatg tagtcttatg caatactctt
     5941 gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc ttacaaggag agaaaaagca
     6001 ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt gccttattag gaaggcaaca
     6061 gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag atattgtatt
     6121 taagtgccta gctcgataca taaacgggtc tctctggtta gaccagatct gagcctggga
     6181 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     6241 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     6301 ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg
     6361 aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc
     6421 gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga
     6481 gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt
     6541 cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg
     6601 gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa
     6661 atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat
     6721 aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa
     6781 gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc
     6841 gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg aattatataa
     6901 atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt
     6961 ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc
     7021 agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt
     7081 gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct
     7141 gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag
     7201 atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac
     7261 cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca
     7321 cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt
     7381 aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa
     7441 atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt
     7501 cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt
     7561 gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag
     7621 gggac
//
