LOCUS       Exported                7314 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pINT-PuroDDHNF4a2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7314)
  AUTHORS   Waldner C, Rempel O, Schutte F, Yanik M, Solomentsew N, Ryffel GU
  TITLE     Double conditional human embryonic kidney cell line based on FLP and
            PhiC31 mediated transgene integration.
  JOURNAL   BMC Res Notes. 2011 Oct 18;4:420.
  PUBMED    22008483
REFERENCE   2  (bases 1 to 7314)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7314
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        3..987
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1022..1040
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1022..1039
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      1072..1081
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1078..1401
                     /codon_start=1
                     /product="destabilization domain that can be stabilized by 
                     Shield1 in the ProteoTuner(TM) system"
                     /label=DD
                     /note="L106P mutant of FKBP12"
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKPE"
     promoter        complement(3467..3484)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3489..3623
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3736..3756)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3736..3754)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3775..3791)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3775..3791)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3788..3810)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3799..3815
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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(3823..3853)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3868..3889
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4006..4023)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4177..4765)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4257..4276)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4936..5796)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5559..5578
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5797..5901)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5927..6382)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6064..6085)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6276..6295
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    6459..6492
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             6493..7089
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     primer_bind     6986..7006
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7113..7247
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 tcgaccatag ccaattcaat atggcgtata tggactcatg ccaattcaat atggtggatc
       61 tggacctgtg ccaattcaat atggcgtata tggactcgtg ccaattcaat atggtggatc
      121 tggaccccag ccaattcaat atggcggact tggcaccatg ccaattcaat atggcggact
      181 tggcactgtg ccaactgggg aggggtctac ttggcacggt gccaagtttg aggaggggtc
      241 ttggccctgt gccaagtccg ccatattgaa ttggcatggt gccaataatg gcggccatat
      301 tggctatatg ccaggatcaa tatataggca atatccaata tggccctatg ccaatatggc
      361 tattggccag gttcaatact atgtattggc cctatgccat atagtattcc atatatgggt
      421 tttcctattg acgtagatag cccctcccaa tgggcggtcc catataccat atatggggct
      481 tcctaatacc gcccatagcc actcccccat tgacgtcaat ggtctctata tatggtcttt
      541 cctattgacg tcatatgggc ggtcctattg acgtatatgg cgcctccccc attgacgtca
      601 attacggtaa atggcccgcc tggctcaatg cccattgacg tcaataggac cacccaccat
      661 tgacgtcaat gggatggctc attgcccatt catatccgtt ctcacgcccc ctattgacgt
      721 caatgacggt aaatggccca cttggcagta catcaatatc tattaatagt aacttggcaa
      781 gtacattact attggaagta cgccagggta cattggcagt actcccattg acgtcaatgg
      841 cggtaaatgg cccgcgatgg ctgccaagta catccccatt gacgtcaatg gggaggggca
      901 atgacgcaaa tgggcgttcc attgacgtaa atgggcggta ggcgtgccta atgggaggtc
      961 tatataagca atgctcgttt agggaaccgc cattctgcct ggggacgtcg gagcaagctt
     1021 gatttaggtg acactataga atacaagcta cttgttcttt ttgcaggatc cgccaccatg
     1081 ggagtgcagg tggaaaccat ctccccagga gacgggcgca ccttccccaa gcgcggccag
     1141 acctgtgtgg tgcactacac cgggatgctt gaagatggaa agaaagtcga ttcctcccgg
     1201 gacagaaaca agccctttaa gtttatgcta ggcaagcagg aggtgatccg aggctgggaa
     1261 gaaggggttg cccagatgag tgtgggtcag agagccaaac tgactatatc tccagattat
     1321 gcctatggtg ccactgggca cccaggcatc atcccaccac atgccactct cgtcttcgat
     1381 gtggagcttc taaaaccgga agaattccga ctctccaaaa ccctcgtcga catggacatg
     1441 gccgactaca gtgctgcact ggacccagcc tacaccaccc tggaatttga gaatgtgcag
     1501 gtgttgacga tgggcaatga cacgtcccca tcagaaggca ccaacctcaa cgcgcccaac
     1561 agcctgggtg tcagcgccct gtgtgccatc tgcggggacc gggccacggg caaacactac
     1621 ggtgcctcga gctgtgacgg ctgcaagggc ttcttccgga ggagcgtgcg gaagaaccac
     1681 atgtactcct gcagatttag ccggcagtgc gtggtggaca aagacaagag gaaccagtgc
     1741 cgctactgca ggctcaagaa atgcttccgg gctggcatga agaaggaagc cgtccagaat
     1801 gagcgggacc ggatcagcac tcgaaggtca agctatgagg acagcagcct gccctccatc
     1861 aatgcgctcc tgcaggcgga ggtcctgtcc cgacagatca cctcccccgt ctccgggatc
     1921 aacggcgaca ttcgggcgaa gaagattgcc agcatcgcag atgtgtgtga gtccatgaag
     1981 gagcagctgc tggttctcgt tgagtgggcc aagtacatcc cagctttctg cgagctcccc
     2041 ctggacgacc aggtggccct gctcagagcc catgctggcg agcacctgct gctcggagcc
     2101 accaagagat ccatggtgtt caaggacgtg ctgctcctag gcaatgacta cattgtccct
     2161 cggcactgcc cggagctggc ggagatgagc cgggtgtcca tacgcatcct tgacgagctg
     2221 gtgctgccct tccaggagct gcagatcgat gacaatgagt atgcctacct caaagccatc
     2281 atcttctttg acccagatgc caaggggctg agcgatccag ggaagatcaa gcggctgcgt
     2341 tcccaggtgc aggtgagctt ggaggactac atcaacgacc gccagtatga ctcgcgtggc
     2401 cgctttggag agctgctgct gctgctgccc accttgcaga gcatcacgtg gcagatgatc
     2461 gagcagatcc agttcatcaa gctcttcggc atggccaaga ttgacaacct gttgcaggag
     2521 atgctgctgg gagggtcccc cagcgatgca ccccatgccc accaccccct gcaccctcac
     2581 ctgatgcagg aacatatggg aaccaacgtc atcgttgcca acacaatgcc cactcacctc
     2641 agcaacggac agatgtgtga gtggccccga cccaggggac aggcagccac ccctgagacc
     2701 ccacagccct caccgccagg tggctcaggg tctgagccct ataagctcct gccgggagcc
     2761 gtcgccacaa tcgtcaagcc cctctctgcc atcccccagc cgaccatcac caagcaggaa
     2821 gttatctagc aagccgctgg ggcttggggg ctccactggc tccccccagc cccctaagag
     2881 agcacctggt gatcacgtgg tcacggcaaa ggaagacgtg atgccaggac cagtcccaga
     2941 gcaggaatgg gaaggatgaa gggcccgaga acatggccta agggccacat cccactgcca
     3001 cccttgacgc cctgctctgg ataacaagac tttgacttgg ggagacctct actgccttgg
     3061 acaacttttc tcatgttgaa gccactgcct tcaccttcac cttcatccat gtccaacccc
     3121 cgacttcatc ccaaaggaca gccgcctgga gatgacttga ggccttactt aaacccagct
     3181 cccttcttcc ctagcctggt gcttctcctc tcctagcccc ggtcatggtg tccagacaga
     3241 gccctgtgag gctgggtcca attgtggcac ttggggcacc ttgctcctcc ttctgctgct
     3301 gcccccacct ctgctgcctc cctctgctgt caccttgctc agccatcccg tcttctccaa
     3361 caccacctct acagaggcca aggaggcctt ggaaacgatt cccccagtca ttctgggaac
     3421 atgttgtaag cactgactgg gaccaggcac caggcagggt ctagaactat agtgagtcgt
     3481 attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa
     3541 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     3601 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     3661 agggggaggt gtgggaggtt ttttaattcg cggccgcggc gccaatgcat tgggcccggt
     3721 acccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata
     3781 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3841 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     3901 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3961 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4021 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4081 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4141 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4201 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4261 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4321 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4381 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4441 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4501 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4561 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     4621 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4681 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4741 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4801 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4861 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4921 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4981 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5041 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5101 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5161 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5221 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5281 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5341 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5401 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5461 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5521 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5581 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5641 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5701 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5761 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5821 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5881 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     5941 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     6001 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     6061 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     6121 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     6181 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     6241 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     6301 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     6361 aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg
     6421 cgatcggtgc gggcctcttc gctattacgc cagtcgaggt gccagggcgt gcccttgggc
     6481 tccccgggcg cgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     6541 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     6601 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     6661 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     6721 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     6781 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     6841 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     6901 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     6961 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     7021 gtcaccgccg acgtcgagtg cccgaaggac cgcgcgacct ggtgcatgac ccgcaagccc
     7081 ggtgcctgag aattcgtgag tcgtattacg tagatccaga catgataaga tacattgatg
     7141 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     7201 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     7261 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa ttcg
//
