LOCUS       pRC0462_pSB700-Neomycin        9136 bp ds-DNA     circular SYN 17-SEP-2022
DEFINITION  Lentiviral vector for expressing U6 sgRNA and CAGGS Cerulean
            fluorescent protein.
ACCESSION
VERSION     .
KEYWORDS    pSB700.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct.
REFERENCE   1  (bases 1 to 9061)
  AUTHORS   Chavez, A, Scheiman, J, Vora, S, Pruitt, BW, Tuttle, M, P, R, Iyer,
            E, Lin, S, Kiani, S, Guzman, CD, Wiegand, DJ, Ter-Ovanesyan, D,
            Braff, JL, Davidsohn, N, Housden, BE, Perrimon, N, Weiss, R, Aach,
            J, Collins, JJ, Church and GM
  TITLE     Highly efficient Cas9-mediated transcriptional programming.
  JOURNAL   Nat Methods. 2015 Mar 2. doi: 10.1038/nmeth.3312.
  PUBMED    25730490
REFERENCE   2  (bases 1 to 9061)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Jun 20, 2017 from SnapGene Console 4.0.0a8
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     LTR             1..634
                     /note="3' long terminal repeat (LTR) from HIV-1"
                     /label="3' LTR"
     source          1..9136
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    681..806
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
                     /label="HIV-1 Psi"
     misc_feature    1303..1536
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to
                     thecytoplasm."
                     /label="RRE"
     misc_feature    2028..2143
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
                     /label="cPPT/CTS"
     PCR_primer      2209..2228
                     /created_by="charir2"
                     /label="IA"
     promoter        2246..2486
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /label="U6 promoter"
     primer_bind     2246..2266
                     /note="Human U6 promoter, forward primer"
                     /label="hU6-F"
     primer_bind     2417..2436
                     /note="Human U6 promoter, forward primer"
                     /label="LKO.1 5'"
     misc_RNA        2516..2591
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /label="g RNA scaffold"
     PCR_primer      complement(2599..2620)
                     /created_by="charir2"
                     /modified_by="charir2"
                     /label="R"
     enhancer        2665..2968
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        2970..3244
                     /label="chicken beta-actin promoter"
     intron          3246..4263
                     /note="chimera between introns from chicken beta-actin and
                      rabbit beta-globin"
                     /label="chimeric intron"
     primer_bind     4187..4209
                     /note="Chimeric intron in CAG promoter, forward primer"
                     /label="pCAGGS-5"
     primer_bind     4271..4290
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
                     /label="pCAG-F"
     gene            4297..5091
                     /created_by="charir2"
                     /label="Neomycin"
     source          <4297..>5091
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    5104..5692
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /label="WPRE"
     primer_bind     complement(5157..5177)
                     /note="WPRE, reverse primer"
                     /label="WPRE-R"
     CDS             complement(5575..5586)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /translation="IEGR"
                     /label="Factor Xa site"
     LTR             5899..6532
                     /note="3' long terminal repeat (LTR) from HIV-1"
                     /label="3' LTR"
     primer_bind     complement(6661..6677)
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /label="M13 rev"
     primer_bind     complement(6661..6677)
                     /note="In lacZ gene. Also called M13-rev"
                     /label="M13 Reverse"
     primer_bind     complement(6674..6696)
                     /note="In lacZ gene"
                     /label="M13/pUC Reverse"
     protein_bind    6685..6701
                     /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"
     promoter        complement(6709..6739)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    6754..6775
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /label="CAP binding site"
     primer_bind     complement(6892..6909)
                     /note="L4440 vector, forward primer"
                     /label="L4440"
     rep_origin      complement(7063..7651)
                     /direction="LEFT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
     primer_bind     complement(7143..7162)
                     /note="pBR322 origin, forward primer"
                     /label="pBR322ori-F"
     CDS             complement(7822..8682)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /label="AmpR"
     primer_bind     8445..8464
                     /note="Ampicillin resistance gene, reverse primer"
                     /label="Amp-R"
     promoter        complement(8683..8787)
                     /gene="bla"
                     /label="AmpR promoter"
     polyA_signal    8835..8969
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(8872..8891)
                     /note="SV40 polyA, reverse primer"
                     /label="SV40pA-R"
     primer_bind     8926..8945
                     /note="SV40 polyA terminator, reverse primer"
                     /label="EBV-rev"
ORIGIN      
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag
     2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2161 acagcagaga tccagtttat cattagtgaa cggatctcga cggtatcgat cacgagacta
     2221 gcctcgagcg gccgccccct tcaccgaggg cctatttccc atgattcctt catatttgca
     2281 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     2341 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     2401 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     2461 ggctttatat atcttgtgga aaggacgaaa caccggagac gattaatgcg tctcggtttt
     2521 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     2581 cgagtcggtg ctttttttga attctcgacc tcgagacaaa tggcagtatt cgtcattagt
     2641 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     2701 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     2761 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     2821 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     2881 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     2941 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
     3001 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
     3061 tgcagcgatg ggggcggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg
     3121 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
     3181 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
     3241 ggcggggagt cgctgcgacg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
     3301 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
     3361 tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg
     3421 cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt
     3481 gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt
     3541 gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg
     3601 gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg
     3661 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc
     3721 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg
     3781 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc
     3841 ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc
     3901 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag
     3961 agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc
     4021 cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
     4081 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc
     4141 tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg
     4201 cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta
     4261 cagctcctgg gcaacgtgct ggttattgtg gccaccatga ttgaacaaga tggattgcac
     4321 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4381 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4441 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     4501 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4561 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4621 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4681 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4741 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4801 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4861 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4921 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4981 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     5041 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttcta aagcgtctgg
     5101 aacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     5161 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     5221 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     5281 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     5341 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     5401 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     5461 ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt tccatggctg
     5521 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     5581 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     5641 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcctggaatt
     5701 aattctgcag tcgagaccta gaaaaacatg gagcaatcac aagtagcaat acagcagcta
     5761 ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt tttccagtca
     5821 cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
     5881 taaaagaaaa gaggggactg gaagggctaa ttcactccca acgaagacaa gatatccttg
     5941 atctgtggat ctaccacaca caaggctact tccctgatta gcagaactac acaccagggc
     6001 caggggtcag atatccactg acctttggat ggtgctacaa gctagtacca gttgagccag
     6061 ataaggtaga agaggccaat aaaggagaga acaccagctt gttacaccct gtgagcctgc
     6121 atgggatgga tgacccggag agagaagtgt tagagtggag gtttgacagc cgcctagcat
     6181 ttcatcacgt ggcccgagag ctgcatccgg agtacttcaa gaactgctga tatcgagctt
     6241 gctacaaggg actttccgct ggggactttc cagggaggcg tggcctgggc gggactgggg
     6301 agtggcgagc cctcagatcc tgcatataag cagctgcttt ttgcctgtac tgggtctctc
     6361 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     6421 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     6481 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt
     6541 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag
     6601 aggccttgac attgctagcg ttttaccgtc gacctctagc tagagcttgg cgtaatcatg
     6661 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     6721 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     6781 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     6841 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     6901 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     6961 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     7021 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     7081 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     7141 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     7201 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     7261 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     7321 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     7381 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     7441 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     7501 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     7561 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     7621 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     7681 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     7741 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     7801 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     7861 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     7921 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     7981 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     8041 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     8101 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     8161 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     8221 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     8281 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     8341 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     8401 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     8461 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     8521 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     8581 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     8641 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     8701 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     8761 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     8821 cggatcggga gatcaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     8881 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     8941 tcatcaatgt atcttatcat gtctggatca actggataac tcaagctaac caaaatcatc
     9001 ccaaacttcc caccccatac cctattacca ctgccaatta cctgtggttt catttactct
     9061 aaacctgtga ttcctctgaa ttattttcat tttaaagaaa ttgtatttgt taaatatgta
     9121 ctacaaactt agtagt
//
