LOCUS       sequence-262579-        3088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains tracrRNA-hU6 sequence for double sgRNA cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3088)
  AUTHORS   Y Wang
  TITLE     Yongming Wang lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3088)
  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            "Unpublished"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: hU6-Sa_tracr
FEATURES             Location/Qualifiers
     source          1..3088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        270..345
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   359..499
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   359..488
                     /label=AAV2 ITR
     rep_origin      574..1029
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(661..680)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     871..892
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1046..1065)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1165..1187
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1225..1243)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1311..1415
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1416..1484,1485..2276)
                     /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(1634..1653)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2447..3035
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2936..2955
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gagaccacgg caggtctcag ttttagtact ctggaaacag aatctactaa
      301 aacaaggcaa aatgccgtgt ttatctcgtc aacttgttgg cgagattttt gcggccgcag
      361 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
      421 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
      481 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
      541 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
      601 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      661 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
      721 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
      781 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
      841 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
      901 tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt
      961 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     1021 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     1081 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     1141 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     1201 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     1261 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     1321 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     1381 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     1441 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     1501 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     1561 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     1621 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     1681 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     1741 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     1801 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     1861 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1921 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     1981 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     2041 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     2101 ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc
     2161 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     2221 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     2281 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     2341 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     2401 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2461 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2521 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2581 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2641 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2701 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2761 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2821 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2881 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2941 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3001 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3061 ggttcctggc cttttgctgg ccttttgc
//
