LOCUS       Exported                7762 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses an Neomycin-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    Lenti-sgNeo/Cre
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7762)
  AUTHORS   Chiou SH, Winters IP, Wang J, Naranjo S, Dudgeon C, Tamburini FB, 
            Brady JJ, Yang D, Gruner BM, Chuang CH, Caswell DR, Zeng H, Chu P, 
            Kim GE, Carpizo DR, Kim SK, Winslow MM
  TITLE     Pancreatic cancer modeling using retrograde viral vector delivery 
            and in vivo CRISPR/Cas9-mediated somatic genome editing.
  JOURNAL   Genes Dev. 2015 Jul 15;29(14):1576-85. doi: 10.1101/gad.264861.115. 
            Epub 2015 Jul 15.
  PUBMED    26178787
REFERENCE   2  (bases 1 to 7762)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7762
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1686..1919
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2104..2148
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2446..2563
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2645..2885
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2645..2665
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2816..2835
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2915..2990
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(3078..3101)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3475..3494
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3546..3566
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3567..4595
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     misc_feature    4651..5239
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4704..4724)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5122..5133)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5242..5258)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5243..5259)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5768..5948
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6010..6598)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6090..6109)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6769..7629)
                     /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"
     primer_bind     7392..7411
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7630..7734)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgcgctga tcttcagacc tggaggagga
     1561 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     1621 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     1681 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     1741 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     1801 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1861 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1921 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1981 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     2041 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     2101 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     2161 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     2221 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     2281 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     2341 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
     2401 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
     2461 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2521 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2581 cagcagagat ccagtttggt tagtaccggg cccgctctag ccctgcagga actgtttaaa
     2641 ctgtgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     2701 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     2761 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     2821 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     2881 aggaccaaac accgtcatgg ctgatgcaat gcgggtttta gagctagaaa tagcaagtta
     2941 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgttt
     3001 ggcgcgccta tggcttaatt aacatggcgg ccgcattcta ccgggtaggg gaggcgcttt
     3061 tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa
     3121 gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct
     3181 ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc
     3241 cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc
     3301 agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc
     3361 agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc
     3421 tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct
     3481 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggacccg
     3541 ggatgcccaa gaagaagagg aaggtgtcca atttactgac cgtacaccaa aatttgcctg
     3601 cattaccggt cgatgcaacg agtgatgagg ttcgcaagaa cctgatggac atgttcaggg
     3661 atcgccaggc gttttctgag catacctgga aaatgcttct gtccgtttgc cggtcgtggg
     3721 cggcatggtg caagttgaat aaccggaaat ggtttcccgc agaacctgaa gatgttcgcg
     3781 attatcttct atatcttcag gcgcgcggtc tggcagtaaa aactatccag caacatttgg
     3841 gccagctaaa catgcttcat cgtcggtccg ggctgccacg accaagtgac agcaatgctg
     3901 tttcactggt tatgcggcgg atccgaaaag aaaacgttga tgccggtgaa cgtgcaaaac
     3961 aggctctagc gttcgaacgc actgatttcg accaggttcg ttcactcatg gaaaatagcg
     4021 atcgctgcca ggatatacgt aatctggcat ttctggggat tgcttataac accctgttac
     4081 gtatagccga aattgccagg atcagggtta aagatatctc acgtactgac ggtgggagaa
     4141 tgttaatcca tattggcaga acgaaaacgc tggttagcac cgcaggtgta gagaaggcac
     4201 ttagcctggg ggtaactaaa ctggtcgagc gatggatttc cgtctctggt gtagctgatg
     4261 atccgaataa ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg ccatctgcca
     4321 ccagccagct atcaactcgc gccctggaag ggatttttga agcaactcat cgattgattt
     4381 acggcgctaa ggatgactct ggtcagagat acctggcctg gtctggacac agtgcccgtg
     4441 tcggagccgc gcgagatatg gcccgcgctg gagtttcaat accggagatc atgcaagctg
     4501 gtggctggac caatgtaaat attgtcatga actatatccg taacctggat agtgaaacag
     4561 gggcaatggt gcgcctgctg gaagatggcg attagccatg tttaagggtt ccggttccac
     4621 taggtacaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     4681 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     4741 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     4801 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     4861 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     4921 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     4981 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     5041 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     5101 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     5161 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     5221 ctttgggccg cctccccgca tcgataccgt cgacctcgat cgagacctag aaaaacatgg
     5281 agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc tagaagcaca
     5341 agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac caatgactta
     5401 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     5461 tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac aaggctactt
     5521 ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga cctttggatg
     5581 gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa
     5641 cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtatt
     5701 agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc tgcatccgga
     5761 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     5821 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     5881 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     5941 ctctagcagc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     6001 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     6061 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6121 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     6181 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6241 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6301 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6361 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6421 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6481 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6541 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6601 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     6661 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6721 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6781 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6841 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6901 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     6961 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     7021 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     7081 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     7141 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     7201 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     7261 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     7321 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     7381 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     7441 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     7501 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7561 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7621 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     7681 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     7741 tccccgaaaa gtgccacctg ac
//
