LOCUS       sequence-490443-        3332 bp    DNA     circular SYN 23-OCT-2025
DEFINITION  sgRNA for INS activation cloned into LsgRNA-MS2 backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3332)
  AUTHORS   Wu Y, Zhong A, Evangelisti A, Sidharta M, Danwei H, Studer L, Zhou T
  TITLE     Leveraging CRISPR activation for rapid assessment of gene editing 
            products in human pluripotent stem cells.
  JOURNAL   Stem Cell Reports. 2025 Apr 25:102499. doi: 
            10.1016/j.stemcr.2025.102499.
  PUBMED    40345204
REFERENCE   2  (bases 1 to 3332)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 23, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Stem Cell 
            Reports. 2025 Apr 25:102499. doi: 10.1016/j.stemcr.2025.102499."
COMMENT     Sequence Label: INS-CRa-Lsg-MS2
FEATURES             Location/Qualifiers
     source          1..3332
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..38)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(18..36)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(57..73)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(57..73)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(70..92)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    81..97
                     /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(105..111,112..129,130..135))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    150..171
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(288..305)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(459..1047)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(539..558)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1218..2009,2010..2078))
                     /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     1841..1860
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2079..2183)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2209..2664)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2346..2367)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2558..2577
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2791..2813
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2805..2822
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2806..2822
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2832..2851
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2832..2850
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2865..3113
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2865..2885
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3036..3055
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3152..3170
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        3222..3240
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
ORIGIN
        1 agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
       61 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
      121 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
      181 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
      241 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
      301 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
      361 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
      421 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
      481 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
      541 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
      601 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
      661 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
      721 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
      781 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
      841 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
      901 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
      961 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     1021 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     1081 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     1141 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     1201 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     1261 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     1321 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     1381 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     1441 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     1501 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     1561 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     1621 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     1681 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     1741 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     1801 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     1861 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     1921 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     1981 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     2041 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     2101 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     2161 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
     2221 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     2281 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     2341 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     2401 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     2461 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     2521 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     2581 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     2641 ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag
     2701 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     2761 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     2821 gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgagggc ctatttccca
     2881 tgattccttc atatttgcat atacgataca aggctgttag agagataatt agaattaatt
     2941 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     3001 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     3061 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgccagca
     3121 ccagggaaat ggtcgtttta gagctaggcc aacatgagga tcacccatgt ctgcagggcc
     3181 tagcaagtta aaataaggct agtccgttat caacttggcc aacatgagga tcacccatgt
     3241 ctgcagggcc aagtggcacc gagtcggtgc ttttttggat ccaagcttgg cgtaactaga
     3301 tcttgagaca aatggcagta ttcatccaca ag
//
