LOCUS       sequence-232186-        3036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression, Transcription regulation, gRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3036)
  AUTHORS   Teng F, Cui T, Feng G, Guo L, Xu K, Gao Q, Li T, Li J, Zhou Q, Li W
  TITLE     Repurposing CRISPR-Cas12b for mammalian genome engineering.
  JOURNAL   Cell Discov. 2018 Nov 27;4:63. doi: 10.1038/s41421-018-0069-3. 
            eCollection 2018.
  PUBMED    30510770
REFERENCE   2  (bases 1 to 3036)
  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 
            Discov."; date: "2018-11-27"; volume: "4:63. doi"; pages: " 
            10.1038/s41421-018-0069-3. eCollection 2018"
COMMENT     Sequence Label: pUC19-U6-AasgRNA3.8.3
FEATURES             Location/Qualifiers
     source          1..3036
                     /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        402..650
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     402..422
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     573..592
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        729..747
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     primer_bind     complement(815..831)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(815..831)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(828..850)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    839..855
                     /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(863..869,870..887,888..893))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    908..929
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1046..1063)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1217..1805)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1297..1316)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1976..2767,2768..2836))
                     /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     2599..2618
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2837..2941)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3009..3027
                     /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 cgagggccta tttcccatga
      421 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
      481 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
      541 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
      601 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gggtctaaag
      661 gacagaattt ttcaacgggt gtgccaatgg ccactttcca ggtggcaaag cccgttgaac
      721 ttcggccaac atgaggatca cccatgtctg cagggccgaa gtggcacaga gaccgagaga
      781 gggtctcatt ttttttaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
      841 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
      901 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
      961 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1021 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     1081 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     1141 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1201 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1261 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1321 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1381 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1441 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     1501 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     1561 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     1621 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     1681 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1741 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1801 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     1861 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     1921 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     1981 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2041 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2101 ctgcaatgat accgcgtgag ccacgctcac cggctccaga tttatcagca ataaaccagc
     2161 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2221 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     2281 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     2341 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     2401 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     2461 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     2521 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     2581 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     2641 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     2701 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     2761 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     2821 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     2881 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     2941 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     3001 cctataaaaa taggcgtatc acgaggccct ttcgtc
//
