LOCUS       Exported                6854 bp ds-DNA     circular SYN 31-MAY-2019
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    pKF-P14MMP2AG-T2APuroR-5h
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6854)
  AUTHORS   Gabor Balazsi
  TITLE     Direct Submission
  JOURNAL   Exported Friday, May 31, 2019 from SnapGene 3.1.4
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6854
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1..233
                     /label=shared by both circuits
                     /note="shared by both circuits"
     promoter        256..570
                     /label=tight TRE promoter
                     /note="tight TRE promoter"
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    264..282
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    300..318
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    335..353
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    371..389
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    407..425
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    442..460
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    478..496
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     514..569
                     /label=PminCMV?
                     /note="PminCMV?"
     TATA_signal     532..538
     TATA_signal     561..567
                     /label=Initiator Element
                     /note="Initiator Element"
     CDS             575..1318
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="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
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             1328..1384
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="P2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1385..2101
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2102..2155
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="T2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2156..2755
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    2812..3036
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3320..3367
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP recombinase from the Saccharomyces cerevisi"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3375..4397
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4527..4648
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4697..4713)
                     /label=M13 rev
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4721..4737
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac repressor encoded by lacI lac operator"
                     /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)."
     promoter        complement(4745..4775)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4790..4811
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="E. coli catabolite activator protein CAP bindin"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5099..5687)
                     /direction=LEFT
                     /label=ori
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5858..6718)
                     /label=ampR (bla)
                     /note="ampR (bla)"
     promoter        complement(6719..6823)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="bla promoter"
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgtcgaggcc
      241 ctttcgtctt cactcgagtt tactccctat cagtgataga gaacgtatgt cgagtttact
      301 ccctatcagt gatagagaac gatgtcgagt ttactcccta tcagtgatag agaacgtatg
      361 tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat
      421 agagaacgta tgtcgagttt atccctatca gtgatagaga acgtatgtcg agtttactcc
      481 ctatcagtga tagagaacgt atgtcgaggt aggcgtgtac ggtgggaggc ctatataagc
      541 agagctcgtt tagtgaaccg tcagatcgcc tggaatgtct agactggaca agagcaaagt
      601 cataaacggc gctctggaat tactcaatgg agtcggtatc gaaggcctga cgacaaggaa
      661 actcgctcaa aagctgggag ttgagcagcc taccctgtac tggcacgtga agaacaagcg
      721 ggccctgctc gatgccctgc caatcgagat gctggacagg catcataccc acttctgccc
      781 cctggaaggc gagtcatggc aagactttct gcggaacaac gccaagtcat tccgctgtgc
      841 tctcctctca catcgcgacg gggctaaagt gcatctcggc acccgcccaa cagagaaaca
      901 gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct ccctggagaa
      961 cgcactgtac gctctgtccg ccgtgggcca ctttacactg ggctgcgtat tggaggaaca
     1021 ggagcatcaa gtagcaaaag aggaaagaga gacacctacc accgattcta tgcccccact
     1081 tctgagacaa gcaattgagc tgttcgaccg gcagggagcc gaacctgcct tccttttcgg
     1141 cctggaacta atcatatgtg gcctggagaa acagctaaag tgcgaaagcg gcgggccggc
     1201 cgacgccctt gacgattttg acttagacat gctcccagcc gatgcccttg acgactttga
     1261 ccttgatatg ctgcctgctg acgctcttga cgattttgac cttgacatgc tccccggggg
     1321 aagcggagct actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg
     1381 acctatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1441 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     1501 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1561 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1621 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1681 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1741 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1801 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1861 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1921 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1981 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     2041 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     2101 ggagggcaga ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gccctatgac
     2161 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     2221 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     2281 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     2341 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     2401 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     2461 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     2521 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     2581 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     2641 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     2701 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaaccgg
     2761 tgcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     2821 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     2881 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     2941 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3001 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3061 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3121 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3181 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3241 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3301 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag
     3361 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     3421 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     3481 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     3541 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     3601 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     3661 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     3721 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     3781 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     3841 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     3901 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     3961 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     4021 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     4081 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     4141 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     4201 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     4261 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     4321 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     4381 cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa
     4441 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4501 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4561 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4621 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4681 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4741 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4801 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4861 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4921 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4981 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5041 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5101 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5161 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5221 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5281 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5341 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5401 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5461 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5521 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5581 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5641 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5701 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5761 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5821 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5881 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5941 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6001 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6061 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6121 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6181 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6241 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6301 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6361 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6421 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6481 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6541 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6601 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6661 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6721 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6781 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6841 tgccacctga cgtc
//
