LOCUS       pCY_gRNA_hPAX7_P        3973 bp ds-DNA     circular     14-SEP-2020
DEFINITION  An empty gRNA expression vector, used to create a gRNA to a
            specific sequence..
ACCESSION   .
VERSION     .
KEYWORDS    gRNA_Cloning Vector
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3914)
  AUTHORS   Mali P, Yang L, Esvelt KM, Aach J, Guell M, Dicarlo JE, Norville
            JE, Church GM
  TITLE     RNA-Guided Human Genome Engineering via Cas9.
  JOURNAL   Science. 2013 Jan 3.
  PUBMED    23287722
REFERENCE   2  (bases 1 to 3914)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 23, 2020 from SnapGene Server 1.1.58
            http://www.snapgene.com
COMMENT     
COMMENT     ApEinfo:methylated:1
FEATURES             Location/Qualifiers
     source          join(1..1091,1155..3973)
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /label=source:synthetic DNA construct
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     380..399
                     /note="pBR322 origin, forward primer"
                     /label=pBR322ori-F
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     633..650
                     /note="L4440 vector, forward primer"
                     /label=L4440
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     protein_bind    767..788
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label=CAP binding site
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     promoter        803..833
                     /note="promoter for the E. coli lac operon"
                     /label=lac promoter
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     protein_bind    841..857
                     /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)."
                     /label=lac operator
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     846..868
                     /note="In lacZ gene"
                     /label=M13/pUC Reverse
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     865..881
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label=M13 rev
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     865..881
                     /note="In lacZ gene. Also called M13-rev"
                     /label=M13 Reverse
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     promoter        899..917
                     /note="promoter for bacteriophage SP6 RNA polymerase"
                     /label=SP6 promoter
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     899..916
                     /note="SP6 promoter, forward primer"
                     /label=SP6
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     protein_bind    1002..1026
                     /gene="mutant version of attB"
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
                     /label=attB2
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     complement(1192..1211)
                     /note="Human U6 promoter, forward primer"
                     /label=LKO.1 5'
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     complement(1362..1382)
                     /note="Human U6 promoter, forward primer"
                     /label=hU6-F
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     complement(1521..1540)
                     /note="T7 promoter, forward primer"
                     /label=T7
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     promoter        complement(1522..1540)
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label=T7 promoter
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     complement(1547..1564)
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21)
                     Forward"
                     /label=M13 Forward
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     complement(1547..1563)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label=M13 fwd
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     complement(1556..1578)
                     /note="In lacZ gene"
                     /label=M13/pUC Forward
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     CDS             1701..2003
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /note="Plasmids containing the ccdB gene cannot be
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     V SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
                     /label=ccdB
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     1882..1901
                     /note="ccdB gene, forward primer"
                     /label=ccdB-fwd
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     CDS             2352..3146
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="confers resistance to neomycin, kanamycin, and G418
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     P
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
                     S
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
                     Q
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
                     A LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
                     /label=NeoR/KanR
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     complement(2406..2425)
                     /note="Neomycin resistance gene, reverse primer"
                     /label=Neo-R
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     primer_bind     3016..3035
                     /note="Neomycin resistance gene, forward primer"
                     /label=Neo-F
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     regulatory      3346..3355
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /label=vertebrate consensus sequence for strong initiatio
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     CDS             3352..3726
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /note="confers resistance to bleomycin, phleomycin, and
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRD
                     D
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWG
                     R EFALRDPAGNCVHFVAEEQD"
                     /label=BleoR
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
     rep_origin      join(3864..3973,1..479)
                     /direction=RIGHT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label=ori
                     /ApEinfo_fwdcolor=pink
                     /ApEinfo_revcolor=pink
                     /ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0
ORIGIN
        1 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
       61 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
      121 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
      181 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
      241 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
      301 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
      361 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
      421 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
      481 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
      541 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
      601 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
      661 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
      721 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      781 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      841 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      901 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      961 ctagtaacgg ccgccagtgt gctggaattc gccctttaat gccaactttg tacaagaaag
     1021 ctgggtctag aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc
     1081 cttattttaa cTTGCTATTT CTAGCTCTAA AACAAACGAG GTCGAGCCGG GGCGGTGTTT
     1141 CGTCCTTTCC ACAAgatata taaagccaag aaatcgaaat actttcaagt tacggtaagc
     1201 atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac
     1261 tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt
     1321 atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc
     1381 tcttcctgcc cgaccttggt accggatcca gtcgactgaa ttggttcctt taaagcctgc
     1441 ttttttgtac aaagggcgaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg
     1501 catctagagg gcccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt
     1561 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     1621 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     1681 tgcgcagcct atacgtacgg cagtttaagg tttacaccta taaaagagag agccgttatc
     1741 gtctgtttgt ggatgtacag agtgatatta ttgacacgcc ggggcgacgg atggtgatcc
     1801 ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc
     1861 atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg
     1921 ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta
     1981 acctgatgtt ctggggaata taaatgtcag gcatgagatt atcaaaaagg atcttcacct
     2041 agatcctttt cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca
     2101 gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca
     2161 gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat
     2221 tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt
     2281 tcttgccgcc aaggatctga tggcgcaggg gatcaagctc tgatcaagag acaggatgag
     2341 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     2401 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     2461 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     2521 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     2581 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     2641 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     2701 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     2761 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     2821 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     2881 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     2941 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     3001 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     3061 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     3121 atcgccttct tgacgagttc ttctgaatta ttaacgctta caatttcctg atgcggtatt
     3181 ttctccttac gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat
     3241 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     3301 agacaataac cctgataaat gcttcaataa tagcacgtga ggagggccac catggccaag
     3361 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg
     3421 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg
     3481 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg
     3541 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc
     3601 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
     3661 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag
     3721 gactgacacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3781 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3841 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3901 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3961 ctttttccga agg    
//
