LOCUS       sequence-224556-        2959 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To construct multiple sgRNA expression vector for CRISPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2959)
  TITLE     multiple sgRNA vectors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 2959)
  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            "Unpublished"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: pUC57kan-T7-gRNA-U6 V2
FEATURES             Location/Qualifiers
     source          1..2959
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        442..460
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        482..557
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        598..846
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     598..618
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     769..788
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    875..896
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1014..1031)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1185..1773)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1265..1284)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1944..2759)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1944..2759)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2667..2686
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2760..2864)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2932..2950
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa
      421 tgcatctaga tatcggatcc ctaatacgac tcactatagg tgagaccgag agagggtctc
      481 agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
      541 tggcaccgag tcggtgcttt ttttaaaggg cccgtcgact gcagaggcta gctcaccgag
      601 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
      661 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
      721 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
      781 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
      841 aacaccgcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
      901 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
      961 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg cggccgccgc ttcctcgctc
     1021 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     1081 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     1141 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     1201 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     1261 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     1321 ctgccgctta ccggatacct gtccgccttt ctcccttctg gaagcgtggc gctttctcat
     1381 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     1441 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     1501 aacccggcaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     1561 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     1621 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     1681 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     1741 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     1801 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     1861 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     1921 tatgagtaaa cttggtctga cagttagaaa aactcatcga gcatcaaatg aaactgcaat
     1981 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     2041 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     2101 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     2161 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     2221 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     2281 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     2341 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     2401 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccagggatc
     2461 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     2521 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     2581 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     2641 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     2701 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     2761 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     2821 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     2881 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     2941 atcacgaggc cctttcgtc
//
