LOCUS       62056_13375        5482 bp DNA     circular SYN 07-JUL-2019
DEFINITION  Cloning vector pKF-P14MM5h, complete sequence.
ACCESSION   MK816965
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5482)
  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 5482)
  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 5482)
  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..5482
                     /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             650..1393
                     /codon_start=1
                     /label=rtTA-Advanced
                     /note="improved tetracycline-controlled transactivator"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     polyA_signal    1440..1664
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    1948..1995
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2003..3022
                     /codon_start=1
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3155..3288
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3325..3341)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3349..3365)
                     /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(3373..3403)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3418..3439)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3727..4315)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4489..5346)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5347..5451)
                     /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 ctggagacgc catccacgct gttttgacct
      601 ccatagaaga caccgggacc gatccagcct ccgcggcccc gaattcacca tgtctagact
      661 ggacaagagc aaagtcataa acggcgctct ggaattactc aatggagtcg gtatcgaagg
      721 cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc tgtactggca
      781 cgtgaagaac aagcgggccc tgctcgatgc cctgccaatc gagatgctgg acaggcatca
      841 tacccacttc tgccccctgg aaggcgagtc atggcaagac tttctgcgga acaacgccaa
      901 gtcattccgc tgtgctctcc tctcacatcg cgacggggct aaagtgcatc tcggcacccg
      961 cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt gtcagcaagg
     1021 cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta cactgggctg
     1081 cgtattggag gaacaggagc atcaagtagc aaaagaggaa agagagacac ctaccaccga
     1141 ttctatgccc ccacttctga gacaagcaat tgagctgttc gaccggcagg gagccgaacc
     1201 tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc taaagtgcga
     1261 aagcggcggg ccggccgacg cccttgacga ttttgactta gacatgctcc cagccgatgc
     1321 ccttgacgac tttgaccttg atatgctgcc tgctgacgct cttgacgatt ttgaccttga
     1381 catgctcccc gggtaactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     1441 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1501 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1561 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1621 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     1681 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     1741 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1801 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1861 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1921 tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga
     1981 aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt
     2041 tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc
     2101 tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc
     2161 cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat
     2221 tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg
     2281 tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc
     2341 ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg
     2401 cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat
     2461 tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt
     2521 cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct
     2581 cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt
     2641 cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
     2701 ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc
     2761 ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact
     2821 ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga
     2881 cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc
     2941 ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
     3001 cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt
     3061 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3121 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     3181 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3241 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     3301 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     3361 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     3421 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3481 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3541 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3601 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3661 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3721 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3781 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3841 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3901 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3961 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4021 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4081 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4141 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     4201 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4261 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4321 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4381 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4441 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4501 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4561 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4621 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4681 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4741 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4801 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4861 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4921 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4981 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5041 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5101 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5161 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5221 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5281 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5341 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5401 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5461 ccgaaaagtg ccacctgacg tc
//
