LOCUS       Exported                8631 bp ds-DNA     circular SYN 08-NOV-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 8631)
  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..8631
                     /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(3316..3355)
                     /label=R1
     misc_feature    3320..3803
                     /label=Donor fragment
                     /note="Donor fragment"
     misc_feature    3320..3397
                     /label=gRNA scaffold
     misc_feature    3320..3391
                     /label=chimeric guide RNA scaffold 1
                     /note="chimeric guide RNA scaffold 1"
     terminator      3392..3397
                     /note="U6 terminator"
     primer_bind     complement(3398..3437)
                     /label=R1
     promoter        3482..3803
                     /label=mU6
                     /note="/dnas_title=mU6"
                     /note="/vntifkey=21"
     misc_feature    3573..3580
                     /label=Octamer motif
                     /note="Octamer motif"
     misc_feature    3735..3754
                     /label=proximal sequence element
                     /note="proximal sequence element"
     primer_bind     3764..3803
                     /label=mF1
     misc_feature    3772..3780
                     /label=TATA-Box
                     /note="TATA-Box"
     primer_bind     complement(3825..3864)
                     /label=R1
     misc_feature    3827..3907
                     /label=gRNA scaffold
     promoter        3923..4422
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4012..4028)
                     /label=Fishing Reverse
     CDS             4462..5058
                     /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             5077..5130
                     /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             5131..5832
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    6193..6417
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(6443..6461)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6482..6498)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6506..6522
                     /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(6530..6560)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6575..6596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(6884..7472)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7643..8503)
                     /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(8504..8608)
                     /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 acaccgaact
     3301 tcattaattc acaccgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     3361 tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt ttagagctag aaatagcaag
     3421 ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa atggctctag
     3481 atctagagat ccgacgccgc catctctagg cccgcgccgg ccccctcgca cagacttgtg
     3541 ggagaagctc ggctactccc ctgccccggt taatttgcat ataatatttc ctagtaacta
     3601 tagaggctta atgtgcgata aaagacagat aatctgttct ttttaatact agctacattt
     3661 tacatgatag gcttggattt ctataagaga tacaaatact aaattattat tttaaaaaac
     3721 agcacaaaag gaaactcacc ctaactgtaa agtaattgtg tgttttgaga ctataaatat
     3781 gcatgcgaga aaagccttgt ttggttccct caatctaact acaagtttta gagctagaaa
     3841 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     3901 tttttttgga tccaattcta ccgggtaggg gaggcgcttt tcccaaggca gtctggagca
     3961 tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg gcctcgcaca
     4021 cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc cttcgcgcca
     4081 ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg
     4141 acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag caccgctgag
     4201 caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc cttcgctttc
     4261 tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc
     4321 ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca aaagcgcacg
     4381 tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgacctgcat ccatctagat
     4441 ctcgagcagc tgaagcttac catgaccgag tacaagccca cggtgcgcct cgccacccgc
     4501 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     4561 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     4621 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     4681 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     4741 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     4801 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     4861 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     4921 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     4981 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     5041 acccgcaagc ccggtgccgg cggcgggtcc ggaggagagg gcagaggaag tctcctaaca
     5101 tgcggtgacg tggaggagaa tcctggccca atgagcgagc tgattaagga gaacatgcac
     5161 atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac atccgagggc
     5221 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct
     5281 ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa gaccttcatc
     5341 aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg cttcacatgg
     5401 gagagagtca ccacatacga ggacgggggc gtgctgaccg ctacccagga caccagcctc
     5461 caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac atccaacggc
     5521 cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgagacgct gtaccccgct
     5581 gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg gagccatctg
     5641 atcgcaaaca tcaagaccac atatagatcc aagaaacccg ctaagaacct caagatgcct
     5701 ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa cgagacctac
     5761 gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa actggggcac
     5821 aagcttaatt gagcggccgc taggtacctt taagaccaat gacttacaag gcagctgtag
     5881 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaaagaa
     5941 gtcaagatct gctttttgcc tgtactgggt ctctctggtt agaccagagt ctctctggtt
     6001 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     6061 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     6121 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtt taaacccgct
     6181 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     6241 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     6301 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     6361 agggggagga ttgggaagtc aatagcaggc atgctgggga tgcggtgggc tctatgggcg
     6421 gccgttaatt aagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     6481 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     6541 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     6601 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6661 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     6721 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     6781 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6841 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6901 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6961 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     7021 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     7081 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     7141 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     7201 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     7261 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     7321 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     7381 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     7441 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     7501 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     7561 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     7621 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     7681 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7741 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     7801 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7861 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7921 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7981 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     8041 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     8101 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     8161 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     8221 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     8281 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     8341 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     8401 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     8461 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     8521 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     8581 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca c
//
