LOCUS       Exported                8632 bp ds-DNA     circular SYN 08-NOV-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pKLV-gRNA-Bcan-Ntrk1_4
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 8632)
  AUTHORS   msquatrito
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Nov 13, 2019 from SnapGene Viewer 5.0.4
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8632
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        715..1018
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     LTR             1236..1416
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1463..1588
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2087..2320
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    2847..2964
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     2955..2979
                     /label=Fishing Forward
     promoter        3047..3287
                     /label=hU6
                     /label=U6 Promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    3213..3237
                     /label=SEQ fishing (U6Fw)
     primer_bind     complement(3317..3356)
                     /label=R1
     misc_feature    3321..3804
                     /label=Donor fragment
                     /note="Donor fragment"
     misc_feature    3321..3398
                     /label=gRNA scaffold
     misc_feature    3321..3392
                     /label=chimeric guide RNA scaffold 1
                     /note="chimeric guide RNA scaffold 1"
     terminator      3393..3398
                     /note="U6 terminator"
     primer_bind     complement(3399..3438)
                     /label=R1
     promoter        3483..3804
                     /label=mU6
                     /note="/dnas_title=mU6"
                     /note="/vntifkey=21"
     misc_feature    3574..3581
                     /label=Octamer motif
                     /note="Octamer motif"
     misc_feature    3736..3755
                     /label=proximal sequence element
                     /note="proximal sequence element"
     primer_bind     3765..3804
                     /label=mF1
     misc_feature    3773..3781
                     /label=TATA-Box
                     /note="TATA-Box"
     primer_bind     complement(3826..3865)
                     /label=R1
     misc_feature    3828..3908
                     /label=gRNA scaffold
     promoter        3924..4423
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4013..4029)
                     /label=Fishing Reverse
     CDS             4463..5059
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     CDS             5078..5131
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5132..5833
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    6194..6418
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(6444..6462)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6483..6499)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6507..6523
                     /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(6531..6561)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6576..6597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(6885..7473)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7644..8504)
                     /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"
     promoter        complement(8505..8609)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgactagtta
      661 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      721 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      781 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      841 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      901 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      961 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     1021 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     1081 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     1141 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     1201 ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct
     1261 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     1321 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     1381 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
     1441 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
     1501 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
     1561 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     1621 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     1681 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     1741 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     1801 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     1861 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     1921 gcaagcggcc ggccgcgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     1981 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     2041 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     2101 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     2161 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     2221 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     2281 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     2341 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     2401 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     2461 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     2521 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     2581 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     2641 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     2701 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     2761 gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg ccaattctgc
     2821 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     2881 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     2941 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg
     3001 ttagtaccgg gccctacgcg ttactcgagc caaggtcggg caggaagagg gcctatttcc
     3061 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa
     3121 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     3181 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     3241 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgaacc
     3301 acacttgggc tcctaagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     3361 atcaacttga aaaagtggca ccgagtcggt gcttttttgt tttagagcta gaaatagcaa
     3421 gttaaaataa ggctagtccg tttttagcgc gtgcgccaat tctgcagaca aatggctcta
     3481 gatctagaga tccgacgccg ccatctctag gcccgcgccg gccccctcgc acagacttgt
     3541 gggagaagct cggctactcc cctgccccgg ttaatttgca tataatattt cctagtaact
     3601 atagaggctt aatgtgcgat aaaagacaga taatctgttc tttttaatac tagctacatt
     3661 ttacatgata ggcttggatt tctataagag atacaaatac taaattatta ttttaaaaaa
     3721 cagcacaaaa ggaaactcac cctaactgta aagtaattgt gtgttttgag actataaata
     3781 tgcatgcgag aaaagccttg tttggccggc tagctgagat tgctagtttt agagctagaa
     3841 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     3901 ctttttttgg atccaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     3961 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
     4021 acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc
     4081 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
     4141 gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga
     4201 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
     4261 ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg
     4321 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac
     4381 gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca tccatctaga
     4441 tctcgagcag ctgaagctta ccatgaccga gtacaagccc acggtgcgcc tcgccacccg
     4501 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     4561 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     4621 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     4681 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     4741 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     4801 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga
     4861 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     4921 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     4981 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     5041 gacccgcaag cccggtgccg gcggcgggtc cggaggagag ggcagaggaa gtctcctaac
     5101 atgcggtgac gtggaggaga atcctggccc aatgagcgag ctgattaagg agaacatgca
     5161 catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca catccgaggg
     5221 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc
     5281 tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca agaccttcat
     5341 caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg gcttcacatg
     5401 ggagagagtc accacatacg aggacggggg cgtgctgacc gctacccagg acaccagcct
     5461 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca catccaacgg
     5521 ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc tgtaccccgc
     5581 tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg ggagccatct
     5641 gatcgcaaac atcaagacca catatagatc caagaaaccc gctaagaacc tcaagatgcc
     5701 tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca acgagaccta
     5761 cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca aactggggca
     5821 caagcttaat tgagcggccg ctaggtacct ttaagaccaa tgacttacaa ggcagctgta
     5881 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaaaga
     5941 agtcaagatc tgctttttgc ctgtactggg tctctctggt tagaccagag tctctctggt
     6001 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     6061 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     6121 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ttaaacccgc
     6181 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     6241 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     6301 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     6361 aagggggagg attgggaagt caatagcagg catgctgggg atgcggtggg ctctatgggc
     6421 ggccgttaat taagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     6481 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     6541 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     6601 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6661 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     6721 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6781 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     6841 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     6901 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     6961 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     7021 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     7081 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     7141 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     7201 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     7261 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     7321 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     7381 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7441 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7501 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     7561 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     7621 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     7681 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     7741 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     7801 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     7861 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     7921 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     7981 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     8041 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     8101 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     8161 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     8221 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     8281 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     8341 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     8401 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     8461 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     8521 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     8581 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc ac
//
