LOCUS       Exported                6234 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pEnt L1L3 EF1a-tTA-2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6234)
  AUTHORS   Hsiao EC, Nguyen TD, Ng JK, Scott MJ, Chang WC, Zahed H, Conklin BR
  TITLE     Constitutive Gs activation using a single-construct 
            tetracycline-inducible expression system in embryonic stem cells and
            mice.
  JOURNAL   Stem Cell Res Ther. 2011 Mar 4. 2(2):11.
  PUBMED    21375737
REFERENCE   2  (bases 1 to 6234)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6234
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      103..189
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      281..308
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     316..339
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    358..457
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    490..523
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        1214..1425
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1438..1706
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             1762..2769
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
                     EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(1835..1854)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     intron          2806..3378
                     /label=-beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     primer_bind     complement(3510..3529)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3575..3630
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3629..3648)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4027..4044)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    complement(4469..4564)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4674..4693)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4685..5494
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4752..4771)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5584..6172
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg
       61 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      121 cagtcggaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      181 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg
      241 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat
      301 ggcctttttg cgtttctaca aactcttcct gttagttagt tacttaagct cgggccccaa
      361 ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga taagcaatgc
      421 ttttttataa tgccaacttt gtacaaaaaa gcaggctggc gccggaacca attcagtcga
      481 cgtttaaaca taacttcgta tagcatacat tatacgaagt tatctcgagc ctcaggaaac
      541 agggtaccag tgaaagtctg caatgaattc gagttggcgc gcctgtcatt ctaaatctct
      601 ctttcagcct aaagcttttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc
      661 gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc
      721 acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc
      781 gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt gggagggggc
      841 tgtccccgtg agctcccagg cgcgcctgtc attctaaatc tctctttcag cctaaagctt
      901 tttccccgta tccccccagg tgtctgcagg ctcaaagagc agcgagaagc gttcagagga
      961 aagcgatccc gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg
     1021 atgcgggggg agcgccggac cggagcggag ccccgggcgg ctcgctgctg ccccctagcg
     1081 ggggagggac gtaattacat ccctgggggc tttggggggg ggctgtcccc gtgagctccc
     1141 aggcgcgcca actcggtacc cggggatcca catatgttaa ttaagatctg cgatcgctcc
     1201 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
     1261 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
     1321 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     1381 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacagctgaa gcttcgaggg
     1441 gctcgcatct ctccttcacg cgcccgccgc cctacctgag gccgccatcc acgccggttg
     1501 agtcgcgttc tgccgcctcc cgcctgtggt gcctcctgaa ctgcgtccgc cgtctaggta
     1561 agtttaaagc tcaggtcgag accgggcctt tgtccggcgc tcccttggag cctacctaga
     1621 ctcagccggc tctccacgct ttgcctgacc ctgcttgctc aactctacgt ctttgtttcg
     1681 ttttctgttc tgcgccgtta cagatccaag ctgtgaccgg cgcctacaag gcctgcaggt
     1741 cgacggatcc ggggaattca tatgtctaga ttagataaaa gtaaagtgat taacagcgca
     1801 ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag
     1861 ctaggtgtag agcagcctac attgtattgg catgtaaaaa ataagcgggc tttgctcgac
     1921 gccttagcca ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa
     1981 agctggcaag attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat
     2041 cgcgatggag caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc
     2101 gaaaatcaat tagccttttt atgccaacaa ggtttttcac tagagaatgc attatatgca
     2161 ctcagcgctg tggggcattt tactttaggt tgcgtattgg aagatcaaga gcatcaagtc
     2221 gctaaagaag aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct
     2281 atcgaattat ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc
     2341 atatgcggat tagaaaaaca acttaaatgt gaaagtgggt ccgcgtacag ccgcgcgcgt
     2401 acgaaaaaca attacgggtc taccatcgag ggcctgctcg atctcccgga cgacgacgcc
     2461 cccgaagagg cggggctggc ggctccgcgc ctgtcctttc tccccgcggg acacacgcgc
     2521 agactgtcga cggccccccc gaccgatgtc agcctggggg acgagctcca cttagacggc
     2581 gaggacgtgg cgatggcgca tgccgacgcg ctagacgatt tcgatctgga catgttgggg
     2641 gacggggatt ccccgggtcc gggatttacc ccccacgact ccgcccccta cggcgctctg
     2701 gatatggccg acttcgagtt tgagcagatg tttaccgatg cccttggaat tgacgagtac
     2761 ggtgggtagg gggcgcgagg atcctctaga gctgagaact tcagggtgag tttggggacc
     2821 cttgattgtt ctttcttttt cgctattgta aaattcatgt tatatggagg gggcaaagtt
     2881 ttcagggtgt tgtttagaat gggaagatgt cccttgtatc accatggacc ctcatgataa
     2941 ttttgtttct ttcactttct actctgttga caaccattgt ctcctcttat tttcttttca
     3001 ttttctgtaa ctttttcgtt aaactttagc ttgcatttgt aacgaatttt taaattcact
     3061 tttgtttatt tgtcagattg taagtacttt ctctaatcac ttttttttca aggcaatcag
     3121 ggtatattat attgtacttc agcacagttt tagagaacaa ttgttataat taaatgataa
     3181 ggtagaatat ttctgcatat aaattctggc tggcgtggaa atattcttat tggtagaaac
     3241 aactacaccc tggtcatcat cctgcctttc tctttatggt tacaatgata tacactgttt
     3301 gagatgagga taaaatactc tgagtccaaa ccgggcccct ctgctaacca tgttcatgcc
     3361 ttcttctttt tcctacagct cctgggcaac gtgctggttg ttgtgctgtc tcatcatttt
     3421 ggcaaagaat tcactcctca ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc
     3481 aatgccctgg ctcacaaata ccactgagat ctttttccct ctgccaaaaa ttatggggac
     3541 atcatgaagc cccttgagca tctgacttct gggtaataaa ggaaatttat tttcattgca
     3601 atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat
     3661 ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccat atgctggctg
     3721 ccatgaacaa aggttggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc
     3781 attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg
     3841 tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt
     3901 tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatgg agatccctcg
     3961 actgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4021 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4081 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4141 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4201 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4261 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4321 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4381 ggcgctttct caattagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4441 aagctgggct gtgcgcggcc gcagatctca actttattat acaaagttgg cattataaaa
     4501 aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt
     4561 atttgatatc ctgcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga
     4621 taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     4681 tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc
     4741 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     4801 tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     4861 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     4921 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     4981 ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     5041 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     5101 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     5161 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     5221 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     5281 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     5341 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     5401 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     5461 gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca
     5521 ttattcagat tgggccccgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5581 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5641 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5701 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     5761 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5821 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5881 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     5941 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6001 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6061 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6121 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6181 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtt
//
