LOCUS       sequence-287979-        3974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mCtnnb1 gRNA expressing vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3974)
  AUTHORS   Andersson-Rolf A, Mustata RC, Merenda A, Kim J, Perera S, Grego T, 
            Andrews K, Tremble K, Silva JC, Fink J, Skarnes WC, Koo BK
  TITLE     One-step generation of conditional and reversible gene knockouts.
  JOURNAL   Nat Methods. 2017 Jan 30. doi: 10.1038/nmeth.4156.
  PUBMED    28135257
REFERENCE   2  (bases 1 to 3974)
  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: "Nat 
            Methods. 2017 Jan 30. doi: 10.1038/nmeth.4156."
COMMENT     Sequence Label: gRNA-mCtnnb1
FEATURES             Location/Qualifiers
     source          1..3974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1009..1027
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1009..1026
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        1176..1424
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1176..1196
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1347..1366
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1446..1521
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1533..1557)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1632..1651)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1633..1651)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1658..1675)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1658..1674)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1667..1689)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1812..2114
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1993..2012
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2463..3257
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2463..3257
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2517..2536)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3127..3146
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3457..3466
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3463..3837
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat ttaggtgaca
     1021 ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca ctagtaacgg
     1081 ccgccagtgt gctggaattc gccctttgta caaaaaagca ggctttaaag gaaccaattc
     1141 agtcgactgg atccggtacc aaggtcgggc aggaagaggg cctatttccc atgattcctt
     1201 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
     1261 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     1321 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     1381 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtacat catttgtatt
     1441 ctgcagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     1501 aaagtggcac cgagtcggtg ctttttttct agacccagct ttcttgtaca aagttggcat
     1561 taaagggcga attctgcaga tatccatcac actggcggcc gctcgagcat gcatctagag
     1621 ggcccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc
     1681 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     1741 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     1801 tatacgtacg gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg
     1861 tggatgtaca gagtgatatt attgacacgc cggggcgacg gatggtgatc cccctggcca
     1921 gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg
     1981 atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg
     2041 aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt
     2101 tctggggaat ataaatgtca ggcatgagat tatcaaaaag gatcttcacc tagatccttt
     2161 tcacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc agctactggg
     2221 ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc agtgggctta
     2281 catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa ttgccagctg
     2341 gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc
     2401 caaggatctg atggcgcagg ggatcaagct ctgatcaaga gacaggatga ggatcgtttc
     2461 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     2521 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     2581 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     2641 tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     2701 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     2761 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     2821 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     2881 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     2941 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg
     3001 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3061 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3121 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     3181 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     3241 ttgacgagtt cttctgaatt attaacgctt acaatttcct gatgcggtat tttctcctta
     3301 cgcatctgtg cggtatttca caccgcatca ggtggcactt ttcggggaaa tgtgcgcgga
     3361 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3421 ccctgataaa tgcttcaata atagcacgtg aggagggcca ccatggccaa gttgaccagt
     3481 gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg
     3541 ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg
     3601 accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg
     3661 tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc
     3721 cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc
     3781 gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac
     3841 gtgctaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3901 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3961 atcaaaggat cttc
//
