LOCUS       sequence-96391-e        6766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cre recombinase and Renilla luciferase from the EFS 
            promoter and one U6-driven sgRNA targeting LacZ. AAV backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6766)
  AUTHORS   Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, 
            Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, 
            Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A,
            Feng G, Sharp PA, Zhang F
  TITLE     CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.
  JOURNAL   Cell. 2014 Sep 24. pii: S0092-8674(14)01163-5. doi: 
            10.1016/j.cell.2014.09.014.
  PUBMED    25263330
REFERENCE   2  (bases 1 to 6766)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2014 
            Sep 24. pii: S0092-8674(14)01163-5. doi: 
            10.1016/j.cell.2014.09.014."
COMMENT     Sequence Label: 
            AAV:ITR-U6-sgRNA(LacZ)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR
FEATURES             Location/Qualifiers
     source          1..6766
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        156..404
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     156..176
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     327..346
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        426..501
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        562..773
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             787..1719
                     /codon_start=1
                     /product="luciferase from the anthozoan coelenterate 
                     Renilla reniformis (sea pansy)"
                     /label=Rluc
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             1735..1791
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1792..1812
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2839..2865
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    2893..3481
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2946..2966)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3364..3375)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3513..3989
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3712..3731)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4029..4169
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4029..4158
                     /label=AAV2 ITR
     rep_origin      4244..4699
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4331..4350)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4541..4562
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4716..4735)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4835..4857
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4895..4913)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4981..5085
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5086..5154,5155..5946)
                     /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     complement(5304..5323)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6117..6705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6606..6625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtgaggg cctatttccc atgattcctt
      181 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
      241 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      301 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      361 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtgcga atacgcccac
      421 gcgatgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
      481 aaagtggcac cgagtcggtg ctttttttct agaggtacca ggtcttgaaa ggagtgggaa
      541 ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
      601 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
      661 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
      721 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggcgtacg
      781 gccaccatga cttcgaaagt ttatgatcca gaacaaagga aacggatgat aactggtccg
      841 cagtggtggg ccagatgtaa acaaatgaat gttcttgatt catttattaa ttattatgat
      901 tcagaaaaac atgcagaaaa tgctgttatt tttttacatg gtaacgcggc ctcttcttat
      961 ttatggcgac atgttgtgcc acatattgag ccagtagcgc ggtgtattat accagacctt
     1021 attggtatgg gcaaatcagg caaatctggt aatggttctt ataggttact tgatcattac
     1081 aaatatctta ctgcatggtt tgaacttctt aatttaccaa agaagatcat ttttgtcggc
     1141 catgattggg gtgcttgttt ggcatttcat tatagctatg agcatcaaga taagatcaaa
     1201 gcaatagttc acgctgaaag tgtagtagat gtgattgaat catgggatga atggcctgat
     1261 attgaagaag atattgcgtt gatcaaatct gaagaaggag aaaaaatggt tttggagaat
     1321 aacttcttcg tggaaaccat gttgccatca aaaatcatga gaaagttaga accagaagaa
     1381 tttgcagcat atcttgaacc attcaaagag aaaggtgaag ttcgtcgtcc aacattatca
     1441 tggcctcgtg aaatcccgtt agtaaaaggt ggtaaacctg acgttgtaca aattgttagg
     1501 aattataatg cttatctacg tgcaagtgat gatttaccaa aaatgtttat tgaatcggac
     1561 ccaggattct tttccaatgc tattgttgaa ggtgccaaga agtttcctaa tactgaattt
     1621 gtcaaagtaa aaggtcttca tttttcgcaa gaagatgcac ctgatgaaat gggaaaatat
     1681 atcaaatcgt tcgttgagcg agttctcaaa aatgaacaag ctagcggaag cggagccact
     1741 aacttctccc tgttgaaaca agcaggggat gtcgaagaga atcccgggcc acccaagaag
     1801 aagaggaagg tgtccaatct cctgactgtt caccagaacc tccctgcgct gccagtagat
     1861 gccactagcg atgaggtcag gaaaaatctc atggatatgt ttagggatag acaggcgttt
     1921 tctgaacaca cctggaaaat gctgcttagc gtgtgccgat cctgggcagc ctggtgtaag
     1981 ctgaacaatc gcaaatggtt ccccgccgag ccggaggacg tgcgcgatta cctgctgtat
     2041 ctccaggcaa gagggctggc tgtcaagact atccagcagc acttgggcca actgaatatg
     2101 ctgcatcgac gcagcgggct cccccggcct agcgattcaa acgcagtctc ccttgttatg
     2161 aggagaatta gaaaggaaaa cgtagatgcg ggtgagaggg ctaagcaggc tctcgctttt
     2221 gagcggactg atttcgacca ggtcagatcc ctgatggaga acagcgatcg gtgccaggac
     2281 atcaggaacc tcgcatttct gggaattgca tataacacac ttctgcgcat agctgagatc
     2341 gcccggatca gagtgaaaga catcagtcga acggacggcg gccggatgct tattcatatt
     2401 ggacgcacaa agacattggt cagcaccgct ggcgttgaaa aggccttgtc cctgggcgta
     2461 acgaagctgg tggaaagatg gatctcagtg tccggcgtgg ctgacgaccc taataattac
     2521 ttgttctgtc gagtgagaaa aaacggagtc gccgcgccct ctgccaccag ccaattgagt
     2581 acacgggccc ttgaagggat ctttgaggca acccaccgac tcatatacgg agccaaggat
     2641 gacagtggcc agaggtatct cgcctggtca ggtcattctg ctagggtggg ggccgcacga
     2701 gacatggcgc gggcaggagt ctccatacca gagattatgc aagctggagg ttggacaaat
     2761 gtgaacatcg ttatgaacta tatccgcaat cttgactctg aaaccggggc catggtgaga
     2821 ctgctcgaag atggtgacta cccatacgat gttccagatt acgcttaaga attcgatatc
     2881 aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     2941 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     3001 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     3061 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     3121 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     3181 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     3241 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     3301 ccttggctgc tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     3361 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     3421 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     3481 catcgatacc gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag
     3541 tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat
     3601 taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg
     3661 tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg
     3721 aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc
     3781 aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc
     3841 tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct
     3901 ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg
     3961 cgtgaaccac tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga
     4021 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4081 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4141 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     4201 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     4261 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4321 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4381 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4441 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4501 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4561 ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     4621 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4681 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4741 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4801 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4861 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     4921 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     4981 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5041 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5101 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5161 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5221 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5281 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5341 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5401 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5461 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5521 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5581 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5641 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5701 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5761 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     5821 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     5881 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     5941 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6001 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6061 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6121 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6181 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6241 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     6301 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6361 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6421 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6481 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6541 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6601 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6661 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6721 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
