LOCUS       sequence-176980-        6752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cre recombinase and Renilla luciferase from the EFS 
            promoter and one U6-driven sgRNA. AAV backbone with SapI spacer for 
            sgRNA cloning..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6752)
  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 6752)
  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(backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR
FEATURES             Location/Qualifiers
     source          1..6752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        412..487
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        548..759
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             773..1705
                     /codon_start=1
                     /product="luciferase from the anthozoan coelenterate 
                     Renilla reniformis (sea pansy)"
                     /label=Rluc
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             1721..1777
                     /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             1778..1798
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2825..2851
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    2879..3467
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2932..2952)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3350..3361)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3499..3975
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3698..3717)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4015..4155
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4015..4144
                     /label=AAV2 ITR
     rep_origin      4230..4685
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4317..4336)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4527..4548
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4702..4721)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4821..4843
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4881..4899)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4967..5071
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5072..5140,5141..5932)
                     /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(5290..5309)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6103..6691
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6592..6611
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accggaagag cgagctcttc tgttttagag
      421 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      481 tcggtgcttt ttttctagag gtaccaggtc ttgaaaggag tgggaattgg ctccggtgcc
      541 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
      601 aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
      661 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
      721 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg cgtacggcca ccatgacttc
      781 gaaagtttat gatccagaac aaaggaaacg gatgataact ggtccgcagt ggtgggccag
      841 atgtaaacaa atgaatgttc ttgattcatt tattaattat tatgattcag aaaaacatgc
      901 agaaaatgct gttatttttt tacatggtaa cgcggcctct tcttatttat ggcgacatgt
      961 tgtgccacat attgagccag tagcgcggtg tattatacca gaccttattg gtatgggcaa
     1021 atcaggcaaa tctggtaatg gttcttatag gttacttgat cattacaaat atcttactgc
     1081 atggtttgaa cttcttaatt taccaaagaa gatcattttt gtcggccatg attggggtgc
     1141 ttgtttggca tttcattata gctatgagca tcaagataag atcaaagcaa tagttcacgc
     1201 tgaaagtgta gtagatgtga ttgaatcatg ggatgaatgg cctgatattg aagaagatat
     1261 tgcgttgatc aaatctgaag aaggagaaaa aatggttttg gagaataact tcttcgtgga
     1321 aaccatgttg ccatcaaaaa tcatgagaaa gttagaacca gaagaatttg cagcatatct
     1381 tgaaccattc aaagagaaag gtgaagttcg tcgtccaaca ttatcatggc ctcgtgaaat
     1441 cccgttagta aaaggtggta aacctgacgt tgtacaaatt gttaggaatt ataatgctta
     1501 tctacgtgca agtgatgatt taccaaaaat gtttattgaa tcggacccag gattcttttc
     1561 caatgctatt gttgaaggtg ccaagaagtt tcctaatact gaatttgtca aagtaaaagg
     1621 tcttcatttt tcgcaagaag atgcacctga tgaaatggga aaatatatca aatcgttcgt
     1681 tgagcgagtt ctcaaaaatg aacaagctag cggaagcgga gccactaact tctccctgtt
     1741 gaaacaagca ggggatgtcg aagagaatcc cgggccaccc aagaagaaga ggaaggtgtc
     1801 caatctcctg actgttcacc agaacctccc tgcgctgcca gtagatgcca ctagcgatga
     1861 ggtcaggaaa aatctcatgg atatgtttag ggatagacag gcgttttctg aacacacctg
     1921 gaaaatgctg cttagcgtgt gccgatcctg ggcagcctgg tgtaagctga acaatcgcaa
     1981 atggttcccc gccgagccgg aggacgtgcg cgattacctg ctgtatctcc aggcaagagg
     2041 gctggctgtc aagactatcc agcagcactt gggccaactg aatatgctgc atcgacgcag
     2101 cgggctcccc cggcctagcg attcaaacgc agtctccctt gttatgagga gaattagaaa
     2161 ggaaaacgta gatgcgggtg agagggctaa gcaggctctc gcttttgagc ggactgattt
     2221 cgaccaggtc agatccctga tggagaacag cgatcggtgc caggacatca ggaacctcgc
     2281 atttctggga attgcatata acacacttct gcgcatagct gagatcgccc ggatcagagt
     2341 gaaagacatc agtcgaacgg acggcggccg gatgcttatt catattggac gcacaaagac
     2401 attggtcagc accgctggcg ttgaaaaggc cttgtccctg ggcgtaacga agctggtgga
     2461 aagatggatc tcagtgtccg gcgtggctga cgaccctaat aattacttgt tctgtcgagt
     2521 gagaaaaaac ggagtcgccg cgccctctgc caccagccaa ttgagtacac gggcccttga
     2581 agggatcttt gaggcaaccc accgactcat atacggagcc aaggatgaca gtggccagag
     2641 gtatctcgcc tggtcaggtc attctgctag ggtgggggcc gcacgagaca tggcgcgggc
     2701 aggagtctcc ataccagaga ttatgcaagc tggaggttgg acaaatgtga acatcgttat
     2761 gaactatatc cgcaatcttg actctgaaac cggggccatg gtgagactgc tcgaagatgg
     2821 tgactaccca tacgatgttc cagattacgc ttaagaattc gatatcaagc ttatcgataa
     2881 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     2941 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3001 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     3061 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     3121 ttggggcatt gccaccacct gtcagctcct ttccggaact ttcgctttcc ccctccctat
     3181 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     3241 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     3301 ctatgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     3361 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     3421 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgagc
     3481 gctgctcgag agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc
     3541 ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt
     3601 ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag
     3661 gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt
     3721 gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg
     3781 cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg
     3841 tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct
     3901 caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct
     3961 cccttccctg tccttctgat tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac
     4021 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     4081 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     4141 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4201 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     4261 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     4321 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4381 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4441 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4501 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4561 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     4621 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     4681 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     4741 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4801 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4861 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4921 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4981 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     5041 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     5101 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     5161 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     5221 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     5281 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     5341 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     5401 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5461 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     5521 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     5581 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     5641 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     5701 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5761 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5821 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5881 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5941 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     6001 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     6061 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6121 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6181 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6241 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6301 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6361 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6421 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6481 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6541 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6601 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6661 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6721 cctggccttt tgctggcctt ttgctcacat gt
//
