LOCUS       sequence-120334-        7840 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentivirus for establishment of doxycycline-inducible cell line 
            expression CloverCP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7840)
  AUTHORS   Akama-Garren EH, Joshi NS, Tammela T, Chang GP, Wagner BL, Lee DY, 
            Rideout Iii WM, Papagiannakopoulos T, Xue W, Jacks T
  TITLE     A Modular Assembly Platform for Rapid Generation of DNA Constructs.
  JOURNAL   Sci Rep. 2016 Feb 18;6:16836. doi: 10.1038/srep16836.
  PUBMED    26887506
REFERENCE   2  (bases 1 to 7840)
  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: "Sci Rep."; 
            date: "2016-02-18"; volume: "6:16836. doi"; pages: " 
            10.1038/srep16836"
COMMENT     Sequence Label: pTRE:CloverCP-EFS:ZeoR
FEATURES             Location/Qualifiers
     source          1..7840
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        618..816
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             834..1014
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1061..1186
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1685..1918
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2103..2147
                     /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"
     misc_feature    2445..2562
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2673..2987
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    2681..2699
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2717..2735
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2752..2770
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2788..2806
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2824..2842
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2859..2877
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2895..2913
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2960..2984
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3044..3053
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3050..3052,3053..3055,3056..3766)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(3095..3116)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3703..3724
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3782..3800)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3783..3800)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             3809..3856
                     /codon_start=1
                     /product="non-ORF yeast peptide conferring 
                     ubiquitin-dependent degradation"
                     /label=hCL1
                     /note="human codon-optimized"
                     /translation="ACKNWFSSLSHFVIHL"
     CDS             3860..3979
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     promoter        4046..4257
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             4302..4676
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     misc_feature    4729..5317
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4782..4802)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5200..5211)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5320..5336)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5321..5337)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5846..6026
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6088..6676)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6168..6187)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6847..7638,7639..7707))
                     /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     7470..7489
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7708..7812)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc
      841 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      901 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      961 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     1021 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     1081 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     1141 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     1201 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     1261 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     1321 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     1381 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     1441 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     1501 aagtaagacc accgcacagc aagcggccgg ccgcgctgat cttcagacct ggaggaggag
     1561 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     1621 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     1681 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     1741 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     1801 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     1861 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     1921 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     1981 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     2041 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     2101 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     2161 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     2221 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     2281 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
     2341 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac
     2401 tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     2461 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     2521 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     2581 agcagagatc cagtttggtt agtaccgggc ccgggtcgac tagtgatcag tgtgagggag
     2641 tgtaaagctg gtttcgacgg tttcgatatg tcgagtttac tccctatcag tgatagagaa
     2701 cgtatgtcga gtttactccc tatcagtgat agagaacgat gtcgagttta ctccctatca
     2761 gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgagtt
     2821 tactccctat cagtgataga gaacgtatgt cgagtttatc cctatcagtg atagagaacg
     2881 tatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgaggtagg cgtgtacggt
     2941 gggaggccta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agaatacgta
     3001 agtcactaac tcgaacgcta gctgtgcgat cgtttttaat taagccacca tggtgagcaa
     3061 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     3121 cggccacaag ttcagcgtcc gcggcgaggg cgagggcgat gccaccaacg gcaagctgac
     3181 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     3241 cttcggctac ggcgtggcct gcttcagccg ctaccccgac cacatgaagc agcacgactt
     3301 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatctctt tcaaggacga
     3361 cggtacctac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     3421 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     3481 caacttcaac agccacaacg tctatatcac ggccgacaag cagaagaacg gcatcaaggc
     3541 taacttcaag atccgccaca acgttgagga cggcagcgtg cagctcgccg accactacca
     3601 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcca
     3661 tcagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     3721 cgtgaccgcc gccgggatta cacatggcat ggacgagctg tacaagtcta gagggcccta
     3781 ttctatagtg tcacctaaat gcaattctgc ttgcaagaac tggttcagta gcttaagcca
     3841 ctttgtgatc caccttaaca gccacggctt ccctcccgag gtggaggagc aggccgccgg
     3901 caccctgccc atgagctgcg cccaggagag cggcatggat agacaccctg ctgcttgcgc
     3961 cagcgccagg atcaacgtct aagcggccgc aaaccgctgt tcctaggaat cccgaggcct
     4021 tcgagtggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     4081 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
     4141 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     4201 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg
     4261 tcgtgacgcg ggacccgaca ttagcgctac agcttaagcg gatggccaag ttgaccagtg
     4321 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     4381 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     4441 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     4501 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
     4561 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
     4621 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgaaaac
     4681 gttgttgttt ggggttgaat tactcttcta gaccgcggcg cgcccgataa tcaacctctg
     4741 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     4801 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     4861 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     4921 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     4981 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     5041 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     5101 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     5161 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     5221 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     5281 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg acctcgatcg
     5341 agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca atgctgattg
     5401 tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac ctcaggtacc
     5461 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     5521 gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc tgtggatcta
     5581 ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag ggatcagata
     5641 tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga aggtagaaga
     5701 agccaatgaa ggagagaaca cccgcttgtt acaccctgtg agcctgcatg ggatggatga
     5761 cccggagaga gaagtattag agtggaggtt tgacagccgc ctagcatttc atcacatggc
     5821 ccgagagctg catccggact gtactgggtc tctctggtta gaccagatct gagcctggga
     5881 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     5941 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     6001 ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa aggccaggaa
     6061 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     6121 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     6181 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6241 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6301 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6361 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6421 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6481 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6541 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6601 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6661 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6721 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6781 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6841 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6901 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6961 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     7021 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     7081 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     7141 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     7201 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7261 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7321 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7381 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7441 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7501 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7561 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7621 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7681 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7741 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7801 aggggttccg cgcacatttc cccgaaaagt gccacctgac
//
