LOCUS       pDOXOFF-(STOP)99xGly-EG 8706 bp ds-DNA     circular     27-APR-2022
DEFINITION  .
COMMENT     cloning strategy: pDOXOFF-o7v1(mr-ori)-HS-EGFP-3xHA cloning
            strategy  notebook: JY2-94, 95 (2 and 3 sequenced JY2-95) derived
            from pDOXOFF-o7v1-HS-EGFP-3xHA drop out ori with AflIII
            (directional sites flanking ori)
FEATURES             Location/Qualifiers
     rep_origin      40..175
                     /label="SV40 ori"
     promoter        175..246
                     /label="AmpR promoter"
     CDS             247..1107
                     /label="AmpR"
     primer          1001..1020
                     /label="RC456"
                     /note="sequence: agccctcccgtatcgtagtt"
     primer          complement(1223..1242)
                     /label="RC457"
                     /note="sequence: tttacggttcctggcctttt"
     misc_feature    complement(1263..1851)
                     /label="ori"
     primer          1811..1830
                     /label="RC454"
                     /note="sequence: tttgcaagcagcagattacg"
     primer          complement(1967..1986)
                     /label="RC455"
                     /note="sequence: agctcactcaaaggcggtaa"
     protein_bind    2154..2175
                     /label="CAP binding site"
     promoter        2190..2220
                     /label="lac promoter"
     protein_bind    2228..2244
                     /label="lac operator"
     primer_bind     2252..2268
                     /label="M13 rev"
     promoter        2289..2307
                     /label="T3 promoter"
     promoter        2335..2561
                     /label="RSV promoter"
     LTR             2562..2742
                     /label="5' LTR (truncated)"
     misc_feature    2789..2914
                     /label="HIV-1 Psi"
     misc_feature    3407..3640
                     /label="RRE"
     CDS             3825..3869
                     /label="gp41 peptide"
     primer          4112..4136
                     /label="QB0226 (pDOXOFF)"
                     /note="sequence: cagtgcaggggaaagaatagtagac"
     misc_feature    4276..4294
                     /label="tetO"
     misc_feature    4312..4330
                     /label="tetO"
     misc_feature    4348..4366
                     /label="tetO"
     misc_feature    4384..4402
                     /label="tetO"
     misc_feature    4420..4438
                     /label="tetO"
     misc_feature    4456..4474
                     /label="tetO"
     misc_feature    4492..4510
                     /label="tetO"
     primer          4550..4568
                     /label="RC426"
                     /note="sequence: agcagagctcgtttagtga"
     primer          complement(4641..4660)
                     /label="pDOXOFF_XhoI/RPTOR_Rvs_2"
                     /note="sequence: cctggccaactgactttacg"
     primer          complement(4668..4683)
                     /label="RC339"
                     /note="sequence: ggcactaggcctggcc"
     None            4690..4695
                     /label="Kozak"
     CDS             4696..5874
                     /label="Translation 4696-5874"
                     /translation="*GLPLPGGVRQRGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGWASTVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGYPYDVPDYAGTGSYPYDVPDYAGSAGYPYDVPDYA*"
     primer          5041..5058
                     /label="EGFP_Fwd"
                     /note="sequence: gtgagcaagggcgaggag"
     misc_feature    5041..5754
                     /label="EGFP"
     primer_bind     complement(5083..5104)
                     /label="EGFP-N"
     primer_bind     complement(5344..5363)
                     /label="EXFP-R"
     primer_bind     5691..5712
                     /label="EGFP-C"
     primer          complement(5735..5754)
                     /label="EGFP_Rvs"
                     /note="sequence: cttgtacagctcgtccatgc"
     misc_feature    5767..5871
                     /label="3xHA"
     primer          5872..5890
                     /label="RC340"
                     /note="sequence: TAGtgaggatccggggttg"
     promoter        5884..6394
                     /label="hPGK promoter"
     primer          complement(5939..5953)
                     /label="RC427"
                     /note="sequence: accacgcccagagca"
     misc_feature    6404..6805
                     /label="BlastR"
     CDS             6806..6859
                     /label="T2A"
     CDS             6860..7606
                     /label="tTA-Advanced"
     CDS             complement(8064..8075)
                     /label="Factor Xa site"
     LTR             8280..8513
                     /label="3' LTR (Delta-U3)"
     polyA_signal    8585..8706
                     /label="SV40 poly(A) signal"
ORIGIN
        1 tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc
       61 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
      121 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctttca
      181 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
      241 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
      301 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
      361 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
      421 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
      481 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
      541 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
      601 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
      661 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
      721 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
      781 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
      841 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
      901 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
      961 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     1021 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     1081 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     1141 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     1201 cACGCGTgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1261 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1321 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1381 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1441 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1501 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1561 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     1621 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     1681 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     1741 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     1801 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     1861 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     1921 gAcatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     1981 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2041 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     2101 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     2161 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     2221 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     2281 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt
     2341 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac
     2401 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct
     2461 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt
     2521 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc
     2581 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     2641 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     2701 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     2761 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     2821 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     2881 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     2941 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     3001 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     3061 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     3121 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     3181 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     3241 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     3301 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     3361 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     3421 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     3481 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     3541 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     3601 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     3661 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     3721 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     3781 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     3841 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     3901 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     3961 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     4021 acctcccaac cCCGAGGGGA CAATTCTCGA CCTCGAGACA AATGGCAGTA TTCATCCACA
     4081 ATTTTAAAAG AAAAGGGGGG ATTGGGGGGT ACAGTGCAGG GGAAAGAATA GTAGACATAA
     4141 TAGCAACAGA CATACAAACT AAAGAATTAC AAAAACAAAT TACAAAAATT CAAAATTTTC
     4201 GGGTTTATTA CAGGGACAGC AGAGATCCAC TTTGGCCGCG AATCGGGATT CGCCCTTAAG
     4261 CTAGCTCGAG TTTACTCCCT ATCAGTGATA GAGAACGTAT GAAGAGTTTA CTCCCTATCA
     4321 GTGATAGAGA ACGTATGCAG ACTTTACTCC CTATCAGTGA TAGAGAACGT ATAAGGAGTT
     4381 TACTCCCTAT CAGTGATAGA GAACGTATGA CCAGTTTACT CCCTATCAGT GATAGAGAAC
     4441 GTATCTACAG TTTACTCCCT ATCAGTGATA GAGAACGTAT ATCCAGTTTA CTCCCTATCA
     4501 GTGATAGAGA ACGTATAAGC TTTAGGCGTG TACGGTGGGC GCCTATAAAA GCAGAGCTCG
     4561 TTTAGTGAAC CGTCAGATCG CCTGGAGCAA TTCCACAACA CTTTTGTCTT ATACCAACTT
     4621 TCCGTACCAC TTCCTACCCT CGTAAAGTCA GTTGGCCAGg cctagttggc caggcctagt
     4681 gccGAATTCg ccaccTAGGG GTTgccgctg ccagggggcg tgcggcagcg cggcggcggc
     4741 ggcggcggcg gcggcggcgg cggcggcggc ggaggcggcg gcggcggcgg cggcggcggc
     4801 ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg gcggcggcgg cggcggcggc
     4861 ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg gcggcggcgg cggcggcggc
     4921 ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg gcggcggcgg cggcggcggc
     4981 ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg gcggcggctg ggccTCGACG
     5041 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     5101 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     5161 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     5221 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     5281 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     5341 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     5401 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     5461 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     5521 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     5581 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     5641 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     5701 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggaggg
     5761 agcggctatc cctatgacgt gcctgattac gccggcacag gatcataccc ctatgatgtg
     5821 cctgactacg ctggcagcgc cggataccct tatgatgtgc ctgattatgc tTAGtgagga
     5881 tccggggttg gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg
     5941 ctctgggcgt ggttccggga aacgcagcgg cgccgaccct gggtctcgca cattcttcac
     6001 gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct
     6061 tcctgctccg cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca
     6121 aacggaagcc gcacgtctca ctagtaccct cgcagacgga cagcgccagg gagcaatggc
     6181 agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag
     6241 cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt
     6301 cctgcccgcg cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc
     6361 cctcgttgac cgaatcaccg acctctctcc ccagcaattc accatggttc ctttgtctca
     6421 agaagaatcc accctcattg aaagagcaac ggctacaatc aacagcatcc ccatctctga
     6481 agactacagc gtcgccagcg cagctctctc tagcgacggc cgcatcttca ctggtgtcaa
     6541 tgtatatcat tttactgggg gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc
     6601 tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt
     6661 gagcccctgc ggacggtgcc gacaggtgct tctcgatctg catcctggga tcaaagccat
     6721 agtgaaggac agtgatggac agccgacggc agttgggatt cgtgaattgc tgccctctgg
     6781 ttatgtgtgg gagggccccg gtgccgaagg tagaggttct ctcctcactt gtggtgatgt
     6841 tgaagaaaac cctggtccaa tgtctagact ggacaagagc aaagtcataa actctgctct
     6901 ggaattactc aatgaagtcg gtatcgaagg cctgacgaca aggaaactcg ctcaaaagct
     6961 gggagttgag cagcctaccc tgtactggca cgtgaagaac aagcgggccc tgctcgatgc
     7021 cctggcaatc gagatgctgg acaggcatca tacccacttc tgccccctgg aaggcgagtc
     7081 atggcaagac tttctgcgga acaacgccaa gtcattccgc tgtgctctcc tctcacatcg
     7141 cgacggggct aaagtgcatc tcggcacccg cccaacagag aaacagtacg aaaccctgga
     7201 aaatcagctc gcgttcctgt gtcagcaagg cttctccctg gagaacgcac tgtacgctct
     7261 gtccgccgtg ggccacttta cactgggctg cgtattggag gatcaggagc atcaagtagc
     7321 aaaagaggaa agagagacac ctaccaccga ttctatgccc ccacttctga gacaagcaat
     7381 tgagctgttc gaccatcagg gagccgaacc tgccttcctt ttcggcctgg aactaatcat
     7441 atgtggcctg gagaaacagc taaagtgcga aagcggcggg ccggccgacg cccttgacga
     7501 ttttgactta gacatgctcc cagccgatgc ccttgacgac tttgaccttg atatgctgcc
     7561 tgctgacgct cttgacgatt ttgaccttga catgctcccc gggtaactaa gtaaggatcg
     7621 atccaagata tcgtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     7681 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     7741 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     7801 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     7861 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     7921 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     7981 cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg
     8041 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     8101 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     8161 ccctttgggc cgcctccccg cctgtttcgc ctcggcgtcc ggactagagg tacctttaag
     8221 accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact
     8281 ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct
     8341 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     8401 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     8461 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag
     8521 ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga
     8581 gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     8641 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     8701 atctta
//