LOCUS       sequence-235323-        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     2..24
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     16..33
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     17..33
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        40..288
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     40..60
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     211..230
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        367..385
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     primer_bind     complement(453..469)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(453..469)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(466..488)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    477..493
                     /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(501..507,508..525,526..531))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    546..567
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(684..701)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(855..1443)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(935..954)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1614..2405,2406..2474))
                     /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     2237..2256
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2475..2579)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2647..2665
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2703..2725)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2825..2844
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tcccagtcac gacgttgtaa aacgacggcc agtgaattcg agggcctatt tcccatgatt
       61 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
      121 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
      181 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
      241 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg gtctaaagga
      301 cagaattttt caacgggtgt gccaatggcc actttccagg tggcaaagcc cgttgaactt
      361 cggccaacat gaggatcacc catgtctgca gggccgaagt ggcacagaga ccgagagagg
      421 gtctcatttt ttttaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
      481 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
      541 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
      601 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
      661 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
      721 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
      781 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
      841 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
      901 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
      961 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     1021 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     1081 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     1141 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     1201 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     1261 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     1321 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     1381 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     1441 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     1501 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     1561 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     1621 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     1681 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1741 gcaatgatac cgcgagagcc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     1801 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     1861 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     1921 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     1981 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     2041 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     2101 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     2161 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     2221 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     2281 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     2341 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     2401 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     2461 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     2521 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     2581 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     2641 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     2701 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     2761 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     2821 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
     2881 agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt
     2941 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     3001 gctgcaaggc gattaagttg ggtaacgcca gggttt
//
