LOCUS       sequence-89574-e        6711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  retroviral bicistronic expression vector for TetR-KRAB and 
            Blasticidin S resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6711)
  AUTHORS   Sigl R, Ploner C, Shivalingaiah G, Kofler R, Geley S
  TITLE     Development of a Multipurpose GATEWAY-Based Lentiviral 
            Tetracycline-Regulated Conditional RNAi System (GLTR).
  JOURNAL   PLoS One. 2014 May 19;9(5):e97764. doi: 
            10.1371/journal.pone.0097764. eCollection 2014.
  PUBMED    24841113
REFERENCE   2  (bases 1 to 6711)
  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: "PLoS One.";
            date: "2014-05-19"; volume: "9(5):e97764. doi"; pages: " 
            10.1371/journal.pone.0097764. eCollection 2014"
COMMENT     Sequence Label: pLIB-TetR-KRAB-IRES-BLAS
FEATURES             Location/Qualifiers
     source          1..6711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    61..606
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(205..222)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     426..445
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     LTR             1079..1672
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(1775..1797)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1883..2023
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1938..1957
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2038..2055)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2209..2797)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2289..2308)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2968..3759,3760..3828))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3591..3610
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3829..3933)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4001..4019
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     LTR             4615..4790
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    4853..5052
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             5253..5669
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     5599..5621
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     5637..5653
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        5675..5693
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5703..6323
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MARLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(5776..5795)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             6324..6344
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6447..6641
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
ORIGIN
        1 gatccaagct tcctagggtc gacgcggccg ccaattcccc ctctccctcc ccccccccta
       61 acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt
      121 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga
      181 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg
      241 tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtacgac cctttgcagg
      301 cagcggaacc cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata
      361 cacctgcaaa ggcggcacaa ccccagtgcc actttgtgag ttggatagtt gtggaaagag
      421 tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc
      481 attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt
      541 taaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg
      601 attatatggc catgaaaaca tttaacattt ctcaacagga tctagaatta gtagaagtag
      661 cgacagagaa gattacaatg ctttatgagg ataataaaca tcatgtggga gcggcaattc
      721 gtacgaaaac aggagaaatc atttcggcag tacatattga agcgtatata ggacgagtaa
      781 ctgtttgtgc agaagccatt gcgattggta gtgcagtttc gaatggacaa aaggattttg
      841 acacgattgt agctgttaga cacccttatt ctgacgaagt agatagaagt attcgagtgg
      901 taagtccttg tggtatgtgt agggagttga tttcagacta tgcaccagat tgttttgtgt
      961 taatagaaat gaatggcaag ttagtcaaaa ctacgattga agaactcatt ccactcaaat
     1021 atacccgaaa ttaatcgata aaataaaaga ttttatttag tctccagaaa aaggggggaa
     1081 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
     1141 ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca gatggaacag
     1201 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
     1261 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
     1321 ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag
     1381 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
     1441 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
     1501 aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg
     1561 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
     1621 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttgggggct
     1681 cgtccgggat cgggagaccc ctgcccaggg accaccgacc caccaccggg aggtaagctg
     1741 gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     1801 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     1861 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     1921 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg
     1981 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     2041 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     2101 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     2161 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2221 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     2281 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2341 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2401 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2461 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2521 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2581 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2641 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2701 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2761 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2821 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     2881 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     2941 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3001 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3061 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     3121 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     3181 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     3241 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc
     3301 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     3361 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     3421 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     3481 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     3541 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc
     3601 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     3661 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     3721 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     3781 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     3841 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     3901 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     3961 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     4021 ctttcgtctt caagaattgc tagcaattaa ttgctagcaa ttcataccag atcaccgaaa
     4081 actgtcctcc aaatgtgtcc ccctcacact cccaaattcg cgggcttctg cctcttagac
     4141 cactctaccc tattccccac actcaccgga gccaaagccg cggcccttcc gtttctttgc
     4201 ttttgaaaga ccccacccgt aggtggcaag ctagcttaag taacgccact ttgcaaggca
     4261 tggaaaaata cataactgag aatagaaaag ttcagatcaa ggtcaggaac aaagaaacag
     4321 ctgaatacca aacaggatat ctgtggtaag cggttcctgc cccggctcag ggccaagaac
     4381 agatgagaca gctgagtgat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg
     4441 gctcggggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag tttctagtga
     4501 atcatcagat gtttccaggg tgccccaagg acctgaaaat gaccctgtac cttatttgaa
     4561 ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ccgctctccg agctcaataa
     4621 aagagcccac aacccctcac tcggcgcgcc agtcttccga tagactgcgt cgcccgggta
     4681 cccgtattcc caataaagcc tcttgctgtt tgcatccgaa tcgtggtctc gctgttcctt
     4741 gggagggtct cctctgagtg attgactacc cacgacgggg gtctttcatt tgggggctcg
     4801 tccgggattt ggagacccct gcccagggac caccgaccca ccaccgggag gtaagctggc
     4861 cagcaactta tctgtgtctg tccgattgtc tagtgtctat gtttgatgtt atgcgcctgc
     4921 gtctgtacta gttagctaac tagctctgta tctggcggac ccgtggtgga actgacgagt
     4981 tctgaacacc cggccgcaac cctgggagac gtcccaggga ctttgggggc cgtttttgtg
     5041 gcccgacctg aggaagggag tcgatgtgga atccgacccc gtcaggatat gtggttctgg
     5101 taggagacga gaacctaaaa cagttcccgc ctccgtctga atttttgctt tcggtttgga
     5161 accgaagccg cgcgtcttgt ctgctgcagc gctgcagcat cgttctgtgt tgtctctgtc
     5221 tgactgtgtt tctgtatttg tctgaaaatt agggccagac tgttaccact cccttaagtt
     5281 tgaccttagg tcactggaaa gatgtcgagc ggatcgctca caaccagtcg gtagatgtca
     5341 agaagagacg ttgggttacc ttctgctctg cagaatggcc aacctttaac gtcggatggc
     5401 cgcgagacgg cacctttaac cgagacctca tcacccaggt taagatcaag gtcttttcac
     5461 ctggcccgca tggacaccca gaccaggtcc cctacatcgt gacctgggaa gccttggctt
     5521 ttgacccccc tccctgggtc aagccctttg tacaccctaa gcctccgcct cctcttcctc
     5581 catccgcccc gtctctcccc cttgaacctc ctcgttcgac cccgcctcga tcctcccttt
     5641 atccagccct cactccttct ctaggcgccg gaattaatac gactcactat aggaattcaa
     5701 ccatggctag attagataaa agtaaagtga ttaacagcgc attagagctg cttaatgagg
     5761 tcggaatcga aggtttaaca acccgtaaac tcgcccagaa gctaggtgta gagcagccta
     5821 cattgtattg gcatgtaaaa aataagcggg ctttgctcga cgccttagcc attgagatgt
     5881 tagataggca ccatactcac ttttgccctt tagaagggga aagctggcaa gattttttac
     5941 gtaataacgc taaaagtttt agatgtgctt tactaagtca tcgcgatgga gcaaaagtac
     6001 atttaggtac acggcctaca gaaaaacagt atgaaactct cgaaaatcaa ttagcctttt
     6061 tatgccaaca aggtttttca ctagagaatg cattatatgc actcagcgct gtggggcatt
     6121 ttactttagg ttgcgtattg gaagatcaag agcatcaagt cgctaaagaa gaaagggaaa
     6181 cacctactac tgatagtatg ccgccattat tacgacaagc tatcgaatta tttgatcacc
     6241 aaggtgcaga gccagccttc ttattcggcc ttgaattgat catatgcgga ttagaaaaac
     6301 aacttaaatg tgaaagtggg tcgccaaaaa agaagagaaa ggtcgacggc ggtggtgctt
     6361 tgtctcctca gcactctgct gtcactcaag gaagtatcat caagaacaag gagggcatgg
     6421 atgctaagtc actaactgcc tggtcccgga cactggtgac cttcaaggat gtatttgtgg
     6481 acttcaccag ggaggagtgg aagctgctgg acactgctca gcagatcgtg tacagaaatg
     6541 tgatgctgga gaactataag aacctggttt ccttgggtta tcagcttact aagccagatg
     6601 tgatcctccg gttggagaag ggagaagagc cctggctggt ggagagagaa attcaccaag
     6661 agacccatcc tgattcagag actgcatttg aaatcaaatc atcagtttaa g
//
