LOCUS       Exported                6047 bp ds-DNA     circular SYN 14-JUN-2016
DEFINITION  Ligation of product* into Exported*.
ACCESSION   .
VERSION     .
KEYWORDS    PN__Pb-gRNA-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6047)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, June 14, 2016 from SnapGene Viewer 3.1.4
            http://www.snapgene.com
COMMENT     Vector molecule:  Exported*
               Fragment ends: NheI and PacI
               Fragment size: 3854
            Insert molecule:  product*
               Fragment ends: PacI and NheI
               Fragment size: 2193
FEATURES             Location/Qualifiers
     source          1..6047
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     603..619
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     LTR             complement(707..941)
                     /note="PB LTR 3'"
     promoter        1182..1422
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        1449..1524
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1576..1693
                     /note="cPPT/CTS"
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1742..2252
                     /note="hPGK promoter"
                     /note="human phosphoglycerate kinase 1 promoter"
     CDS             2274..2873
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /note="PuroR"
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    3020..3141
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     LTR             complement(3398..3710)
                     /note="PB LTR 5'"
     promoter        complement(3856..3874)
                     /note="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3895..3911)
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3919..3935
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="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(3943..3973)
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    3988..4009
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4297..4885)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5056..5916)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5917..6021)
                     /gene="bla"
                     /note="AmpR promoter"
ORIGIN
        1 ggaaattgta aacgttaata 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 gggcgcgtcg cgccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcgg
      661 tattcacgac agcaggctga ataataaaaa aattagaaac tattatttaa ccctagaaag
      721 ataatcatat tgtgacgtac gttaaagata atcatgcgta aaattgacgc atgtgtttta
      781 tcggtctgta tatcgaggtt tatttattaa tttgaataga tattaagttt tattatattt
      841 acacttacat actaataata aattcaacaa acaatttatt tatgtttatt tatttattaa
      901 aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa aacttttaaa cattctctct
      961 tttacaaaaa taaacttatt ttgtacttta aaaacagtca tgttgtatta taaaataagt
     1021 aattagctta acctatacat aatagaaaca aattatactt attagtcagt cagaaacaac
     1081 tttggcacat atcaatatta tgctctcgtt aattaagatt agtgaacgga tctcgacggt
     1141 atcgatcacg agactagcct cgagcggccg cccccttcac cgagggccta tttcccatga
     1201 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
     1261 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     1321 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     1381 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gggtcttcga
     1441 gaagacctgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     1501 gaaaaagtgg caccgagtcg gtgctttttt taaagaattc tcgacctcga gacaaatggc
     1561 agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag
     1621 aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa
     1681 aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc cgcggctcga
     1741 gggggttggg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct
     1801 ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg gactcgcaca ttcttcacgt
     1861 ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc
     1921 ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa
     1981 cggaagccgc acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag
     2041 cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca
     2101 gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc
     2161 tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc
     2221 tcgttgaccg aatcaccgac ctctctcccc agggggatcc accggagctt accatgaccg
     2281 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     2341 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     2401 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     2461 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     2521 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     2581 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     2641 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     2701 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggaaacct
     2761 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     2821 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc
     2881 cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg gtacctttaa
     2941 gaccaatgac ttacaaggca gctgtagatc ttagccactt tctagagtcg gggcggccgg
     3001 ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     3061 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     3121 ataagctgca ataaacaagt tgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3181 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3241 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3301 gtaaagctag ccaacaagct cgtcatcgct ttgcagaaga gcagagagga tatgctcatc
     3361 gtctaaagaa ctacccattt tattatatat tagtcacgat atctataaca agaaaatata
     3421 tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt
     3481 aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata
     3541 gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc
     3601 gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata
     3661 tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa aaaagatttg
     3721 cgctttactc gacctaaact ttaaacacgt catagaatct tcgtttgaca aaaaccacat
     3781 tgtggccaag ctgtgtgacg cgacgcgcgc taaagaatgg caaaccaagt cgcgcgaggt
     3841 acccagcttt tgttcccttt agtgagggtt aattccgagc ttggcgtaat catggtcata
     3901 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3961 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4021 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4081 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4141 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4201 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4261 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4321 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4381 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4441 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4501 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4561 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4621 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4681 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     4741 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4801 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4861 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4921 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4981 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5041 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5101 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5161 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5221 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5281 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5341 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5401 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5461 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5521 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5581 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5641 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5701 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5761 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5821 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5881 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5941 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6001 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctg
//
