LOCUS       sequence-68743-e        2526 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  in vitro transcription of mitfa gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2526)
  AUTHORS   Jao LE, Wente SR, Chen W
  TITLE     Efficient multiplex biallelic zebrafish genome editing using a 
            CRISPR nuclease system.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Aug 5.
  PUBMED    23918387
REFERENCE   2  (bases 1 to 2526)
  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: "Proc Natl 
            Acad Sci U S A. 2013 Aug 5."
COMMENT     Sequence Label: pT7mitfagRNA
FEATURES             Location/Qualifiers
     source          1..2526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     180..202
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     194..211
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     195..211
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        complement(239..314)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(332..351)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(333..351)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(375..391)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(375..391)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(388..410)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    399..415
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(423..429,430..447,448..453))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    468..489
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(606..623)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(777..1365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(857..876)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1536..2327,2328..2396))
                     /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     2159..2178
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2397..2501)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc
       61 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
      121 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
      181 ccagtcacga cgttgtaaaa cgacggccag tgaattcgag ctcggtaccc ggggatccgc
      241 accgactcgg tgccactttt tcaagttgat aacggactag ccttatttta acttgctatt
      301 tctagctcta aaacgccgag cacggcatga cccctatagt gagtcgtatt aatttcaagc
      361 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
      421 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
      481 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
      541 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
      601 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
      661 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
      721 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
      781 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
      841 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
      901 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
      961 gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     1021 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     1081 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     1141 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     1201 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     1261 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     1321 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     1381 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     1441 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     1501 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     1561 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     1621 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     1681 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1741 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1801 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     1861 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     1921 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     1981 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     2041 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     2101 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     2161 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     2221 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     2281 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     2341 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     2401 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     2461 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     2521 ccacct
//
