LOCUS       Exported                5910 bp ds-DNA     circular SYN 04-MAY-2019
DEFINITION  Ligation of Fragment 2 into pDonorJBGK TagBFP Hras WPRE loxp fix 
            040813*.
ACCESSION   .
VERSION     .
KEYWORDS    pDonorJBGK TagBFP2 3Flag cyto WPRE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5910)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sunday, Jun 30, 2019 from SnapGene 4.3.10
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5910
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     polyA_signal    32..493
                     /label=PGK poly(A) signal
                     /note="mouse phosphoglycerate kinase 1 polyadenylation 
                     signal"
     misc_feature    37..254
                     /label=pgk polyA'
     misc_feature    66..71
                     /label=polyA
     misc_feature    66..71
                     /label=polya
     misc_feature    255..498
                     /label=''pgk polyA
     misc_feature    535..1294
                     /label=Sv40 trimer
     misc_feature    535..593
                     /label=trimer1 start
     polyA_signal    641..775
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    672..677
                     /label=polyA
     misc_feature    701..706
                     /label=polyA
     misc_feature    789..834
                     /label=trimer2
     polyA_signal    895..1029
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    926..931
                     /label=polyA
     misc_feature    955..960
                     /label=polyA
     misc_feature    1043..1088
                     /label=Trimer3
     polyA_signal    1149..1283
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    1180..1185
                     /label=polyA
     misc_feature    1209..1214
                     /label=polyA
     protein_bind    1312..1345
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    1312..1345
                     /label=loxP
     misc_feature    1312..1345
                     /label=LoxP
     misc_feature    1325..1332
                     /label=middle site orientation
     primer_bind     complement(1380..1396)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1434..2132
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    1434..2122
                     /label=TagBFP
     misc_feature    1710..1729
                     /label=sgRNA-1
     misc_feature    1730..1732
                     /label=PAM
     misc_feature    1835..1854
                     /label=sgrna 2
     misc_feature    1953..1954
                     /label=i174a
     misc_feature    2142..2204
                     /label=3flag
     misc_feature    2230..2818
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     misc_feature    2230..2817
                     /label=WPRE
     CDS             complement(2701..2712)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2835..3336
                     /label=Rb Beta Globin PolyA
     misc_feature    2835..3336
                     /label=rabbit beta-globin polyadenylation
                     /note="rabbit beta-globin polyadenylation"
     polyA_site      2968..3023
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     misc_feature    2968..2973
                     /label=polyA
     protein_bind    3361..3394
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    3361..3394
                     /label=FRT site
     primer_bind     complement(3413..3429)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3437..3453
                     /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(3461..3491)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3506..3527
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3586..3781
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3632..3767
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     misc_feature    3786..3905
                     /label=SV40 polyA
     polyA_signal    3787..3921
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    3817..3823
                     /label=polya
     misc_feature    3847..3852
                     /label=polyA
     rep_origin      complement(4160..4748)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4919..5779)
                     /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"
     misc_feature    complement(4919..5779)
                     /label=beta lactamase
     promoter        complement(5780..5884)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    5821..5849
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagtggatcc gctgtaagtc tgcagaaatt gatgatctat
       61 taaacaataa agatgtccac taaaatggaa gtttttcctg tcatactttg ttaagaaggg
      121 tgagaacaga gtacctacat tttgaatgga aggattggag ctacgggggt gggggtgggg
      181 tgggattaga taaatgcctg ctctttactg aaggctcttt actattgctt tatgataatg
      241 tttcatagtt ggatatcata atttaaacaa gcaaaaccaa attaagggcc agctcattcc
      301 tcccactcat gatctataga tctatagatc tctcgtggga tcattgtttt tctcttgatt
      361 cccactttgt ggttctaagt actgtggttt ccaaatgtgt cagtttcata gcctgaagaa
      421 cgagatcagc agcctctgtt ccacatacac ttcattctca gtattgtttt gccaagttct
      481 aattccatca gaagcttgca gatctgcgac tctagaggat ctgcgactct agaggatcat
      541 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
      601 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
      661 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
      721 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatctgcga
      781 ctctagagga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
      841 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
      901 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
      961 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     1021 gtctggatct gcgactctag aggatcataa tcagccatac cacatttgta gaggttttac
     1081 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     1141 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     1201 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     1261 atgtatctta tcatgtctgg atccccatca agctgatccg gaacccttaa tataacttcg
     1321 tatagcatac attatacgaa gttattaggt ccctcgacct gcagcccaag ctagcttatc
     1381 gataccgtcg acctcgaggg ggggaattct gcagtcgagg taccgccgcc accatgagcg
     1441 agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg gacaaccatc
     1501 acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa
     1561 tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct actagcttcc
     1621 tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc ttcaagcagt
     1681 ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga
     1741 ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag
     1801 gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct
     1861 tcaccgagac gctgtacccc gctgacggcg gcctggaagg cagaaacgac atggccctga
     1921 agctcgtggg cgggagccat ctgatcgcaa acgccaagac cacatataga tccaagaaac
     1981 ccgctaagaa cctcaagatg cctggcgtct actatgtgga ctacagactg gaaagaatca
     2041 aggaggccaa caacgagacc tacgtcgagc agcacgaggt ggcagtggcc agatactgcg
     2101 acctccctag caaactgggg cacaagctta attccggact cgactacaaa gaccatgacg
     2161 gtgattataa agatcatgac atcgactaca aggatgacga tgactaagcg gccgcactcc
     2221 tcaggtgcta atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     2281 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     2341 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     2401 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     2461 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     2521 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     2581 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     2641 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     2701 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     2761 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcag
     2821 gccgcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     2881 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     2941 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     3001 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     3061 atcagaatga gtatttggtt tagagtttgg caacatatgc catatgctgg ctgccatgaa
     3121 caaaggtggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     3181 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     3241 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     3301 tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct cgacctgcag
     3361 gaagttccta ttctctagaa agtataggaa cttcaagctt ggcgtaatca tggtcatagc
     3421 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3481 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3541 cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc
     3601 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3661 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3721 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3781 aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3841 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3901 gtatcttatc atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3961 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4021 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4081 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4141 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4201 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4261 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4321 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
     4381 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4441 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4501 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4561 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     4621 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4681 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4741 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4801 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4861 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4921 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4981 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5041 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5101 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5161 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5221 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5281 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5341 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5401 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5461 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5521 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5581 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5641 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5701 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5761 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5821 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5881 cgcgcacatt tccccgaaaa gtgccacctg
//
