LOCUS       62056_13380        6855 bp DNA     circular SYN 07-JUL-2019
DEFINITION  Cloning vector pKF-P14MMP2AG-T2APuroR-5h, complete sequence.
ACCESSION   MK816962
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6855)
  AUTHORS   Farquhar KS, Charlebois DA, Szenk M, Cohen J, Nevozhay D, Balazsi G.
  TITLE     Role of network-mediated stochasticity in mammalian drug resistance
  JOURNAL   Nat Commun 10 (1), 2766 (2019)
  PUBMED    31235692
REFERENCE   2  (bases 1 to 6855)
  AUTHORS   Farquhar KS, Charlebois DA, Szenk M, Cohen J, Nevozhay D, Balazsi G.
  TITLE     Direct Submission
  JOURNAL   Submitted (19-APR-2019) Laufer Center for Physical and Quantitative 
            Biology, Stony Brook University, 100 Nicolls Road, Stony Brook, NY 
            11790, USA
REFERENCE   3  (bases 1 to 6855)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun"; date: "2019"; volume: "10"; issue: "1"; pages: "2766"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (19-APR-2019) Laufer Center for Physical and Quantitative Biology, 
            Stony Brook University, 100 Nicolls Road, Stony Brook, NY 11790, 
            USA"
FEATURES             Location/Qualifiers
     source          1..6855
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        257..571
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     CDS             576..1319
                     /label=rtTA-Advanced
                     /note="improved tetracycline-controlled transactivator"
     misc_feature    1329..1385
                     /note="eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides; P2A; from porcine teschovirus 1 polyprotein"
     CDS             1386..2102
                     /label=EGFP
                     /note="enhanced GFP"
     misc_feature    2103..2156
                     /note="eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides; T2A; from Thosea asigna virus capsid protein"
     misc_feature    2157..2756
                     /note="confers resistance to puromycin; PuroR; puromycin 
                     N-acetyltransferase"
     polyA_signal    2813..3037
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3321..3368
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3376..4395
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
     polyA_signal    4528..4661
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4698..4714)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4722..4738)
                     /label=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(4746..4776)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4791..4812)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5100..5688)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5862..6719)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6720..6824)
                     /label=AmpR promoter
ORIGIN
        1 ggacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
       61 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
      121 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg
      181 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgtcgaggc
      241 cctttcgtct tcactcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttac
      301 tccctatcag tgatagagaa cgatgtcgag tttactccct atcagtgata gagaacgtat
      361 gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga
      421 tagagaacgt atgtcgagtt tatccctatc agtgatagag aacgtatgtc gagtttactc
      481 cctatcagtg atagagaacg tatgtcgagg taggcgtgta cggtgggagg cctatataag
      541 cagagctcgt ttagtgaacc gtcagatcgc ctggaatgtc tagactggac aagagcaaag
      601 tcataaacgg cgctctggaa ttactcaatg gagtcggtat cgaaggcctg acgacaagga
      661 aactcgctca aaagctggga gttgagcagc ctaccctgta ctggcacgtg aagaacaagc
      721 gggccctgct cgatgccctg ccaatcgaga tgctggacag gcatcatacc cacttctgcc
      781 ccctggaagg cgagtcatgg caagactttc tgcggaacaa cgccaagtca ttccgctgtg
      841 ctctcctctc acatcgcgac ggggctaaag tgcatctcgg cacccgccca acagagaaac
      901 agtacgaaac cctggaaaat cagctcgcgt tcctgtgtca gcaaggcttc tccctggaga
      961 acgcactgta cgctctgtcc gccgtgggcc actttacact gggctgcgta ttggaggaac
     1021 aggagcatca agtagcaaaa gaggaaagag agacacctac caccgattct atgcccccac
     1081 ttctgagaca agcaattgag ctgttcgacc ggcagggagc cgaacctgcc ttccttttcg
     1141 gcctggaact aatcatatgt ggcctggaga aacagctaaa gtgcgaaagc ggcgggccgg
     1201 ccgacgccct tgacgatttt gacttagaca tgctcccagc cgatgccctt gacgactttg
     1261 accttgatat gctgcctgct gacgctcttg acgattttga ccttgacatg ctccccgggg
     1321 gaagcggagc tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg
     1381 gacctatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1441 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1501 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1561 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1621 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1681 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1741 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1801 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1861 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1921 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1981 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     2041 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2101 aggagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc ggccctatga
     2161 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     2221 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     2281 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     2341 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     2401 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     2461 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     2521 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     2581 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     2641 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     2701 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaaccg
     2761 gtgcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct
     2821 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     2881 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     2941 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3001 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     3061 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3121 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3181 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3241 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3301 ggtgatggtt cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata
     3361 ggaacttcct tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc
     3421 gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct
     3481 ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt
     3541 ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa
     3601 gtgcttgaca ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag
     3661 ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg
     3721 gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc
     3781 ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat
     3841 ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag
     3901 gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac
     3961 gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac
     4021 tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg
     4081 ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt
     4141 gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag
     4201 agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc
     4261 gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc
     4321 tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt
     4381 ccgagggcaa aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa
     4441 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4501 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4561 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4621 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     4681 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4741 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4801 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4861 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4921 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4981 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5041 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5101 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5161 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5221 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5281 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5341 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5401 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5461 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5521 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5581 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5641 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5701 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5761 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5821 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5881 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5941 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6001 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6061 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6121 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6181 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6241 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6301 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6361 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6421 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6481 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6541 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6601 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6661 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6721 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6781 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6841 gtgccacctg acgtc
//
