LOCUS       sequence-258601-        5586 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For loss-of-function experiments in cardiomyocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5586)
  TITLE     CASAAV single gRNA vector
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5586)
  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: 
            "Unpublished"
COMMENT     Sequence Label: AAV-U6-gRNA-TnT-Cre
FEATURES             Location/Qualifiers
     source          1..5586
                     /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"
     intron          1039..1171
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1154..1173
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1216..1234
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1246..1266
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2333..2809
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2532..2551)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2849..2989
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2849..2978
                     /label=AAV2 ITR
     rep_origin      3064..3519
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3151..3170)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3361..3382
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3536..3555)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3655..3677
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3715..3733)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3801..3905
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3906..3974,3975..4766)
                     /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(4124..4143)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4937..5525
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5426..5445
                     /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 ttttctagtg ggataaaagc agtctgggct ttcacatgac agcatctggg
      541 gctgcggcag agggtcgggt ccgaagcgct gccttatcag cgtccccagc cctgggaggt
      601 gacagctggc tggcttgtgt cagcccctcg ggcactcacg tatctccgtc cgacgggttt
      661 aaaatagcaa aactctgagg ccacacaata gcttgggctt atatgggctc ctgtggggga
      721 agggggagca cggagggggc cggggccgct gctgccaaaa tagcagctca caagtgttgc
      781 attcctctct gggcgccggg cacattcctg ctggctctgc ccgccccggg gtgggcgccg
      841 gggggacctt aaagcctctg ccccccaagg agcccttccc agacagccgc cggcacccac
      901 cgctccgtgg gacgatcccc gaagctctag agctttattg cggtagttta tcacagttaa
      961 attgctaacg cagtcagtgc ttctgacaca acagtctcga acttaagctg cagaagttgg
     1021 tcgtgaggca ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag
     1081 aaactgggct tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt
     1141 actgacatcc actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct
     1201 taaggctaga gtacttaata cgactcacta taggctagca tggtgcccaa gaagaagagg
     1261 aaagtctcca acctgctgac tgtgcaccaa aacctgcctg ccctccctgt ggatgccacc
     1321 tctgatgaag tcaggaagaa cctgatggac atgttcaggg acaggcaggc cttctctgaa
     1381 cacacctgga agatgctcct gtctgtgtgc agatcctggg ctgcctggtg caagctgaac
     1441 aacaggaaat ggttccctgc tgaacctgag gatgtgaggg actacctcct gtacctgcaa
     1501 gccagaggcc tggctgtgaa gaccatccaa cagcacctgg gccagctcaa catgctgcac
     1561 aggagatctg gcctgcctcg cccttctgac tccaatgctg tgtccctggt gatgaggaga
     1621 atcagaaagg agaatgtgga tgctggggag agagccaagc aggccctggc ctttgaacgc
     1681 actgactttg accaagtcag atccctgatg gagaactctg acagatgcca ggacatcagg
     1741 aacctggcct tcctgggcat tgcctacaac accctgctgc gcattgccga aattgccaga
     1801 atcagagtga aggacatctc ccgcaccgat ggtgggagaa tgctgatcca cattggcagg
     1861 accaagaccc tggtgtccac agctggtgtg gagaaggccc tgtccctggg ggttaccaag
     1921 ctggtggaga gatggatctc tgtgtctggt gtggctgatg accccaacaa ctacctgttc
     1981 tgccgggtca gaaagaatgg tgtggctgcc ccttctgcca cctcccaact gtccacccgg
     2041 gccctggaag ggatctttga ggccacccac cgcctgatct atggtgccaa ggatgactct
     2101 gggcagagat acctggcctg gtctggccac tctgccagag tgggtgctgc cagggacatg
     2161 gccagggctg gtgtgtccat ccctgaaatc atgcaggctg gtggctggac caatgtgaac
     2221 attgtgatga actacatcag aaacctggac tctgagactg gggccatggt gaggctgctc
     2281 gaggatgggg actgaaagct tatcgatacc gagcgctgct cgagagatct acgggtggca
     2341 tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac
     2401 cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa
     2461 tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag
     2521 ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt ggctcactgc
     2581 aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt tgttgggatt
     2641 ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac ggggtttcac
     2701 catattggcc aggctggtct ccaactccta atctcaggtg atctacccac cttggcctcc
     2761 caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc tgattttgta
     2821 ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc
     2881 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     2941 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     3001 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     3061 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3121 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3181 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3241 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     3301 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3361 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     3421 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     3481 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     3541 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     3601 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3661 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     3721 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     3781 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3841 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3901 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     3961 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4021 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4081 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4141 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4201 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4261 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4321 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4381 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4441 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4501 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4561 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4621 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4681 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4741 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     4801 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     4861 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4921 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4981 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5041 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     5101 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5161 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5221 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5281 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5341 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5401 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5461 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5521 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5581 acatgt
//
