LOCUS       sequence-476552-        5399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses guide#3 to boost the expression of mouse Plxnb2, via 
            CRISPRa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5399)
  AUTHORS   Borrelli C, Roberts M, Eletto D, Hussherr MD, Fazilaty H, Valenta T,
            Lafzi A, Kretz JA, Guido Vinzoni E, Karakatsani A, Adivarahan S, 
            Mannhart A, Kimura S, Meijs A, Baccouche Mhamedi F, Acar IE, Handler
            K, Ficht X, Platt RJ, Piscuoglio S, Moor AE
  TITLE     In vivo interaction screening reveals liver-derived constraints to 
            metastasis.
  JOURNAL   Nature. 2024 Aug;632(8024):411-418. doi: 10.1038/s41586-024-07715-3.
            Epub 2024 Jul 24.
  PUBMED    39048831
REFERENCE   2  (bases 1 to 5399)
  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"; doi: 
            "10.1038/s41586-024-07715-3"; journalName: "Nature"; date: 
            "2024-08"; volume: "632"; issue: "8024"; pages: "411-418"
COMMENT     Sequence Label: sgPlxnb2-3 in pT4-U6-sgRNA-CMV-EGFP
FEATURES             Location/Qualifiers
     source          1..5399
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     263..285
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     277..294
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     278..294
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     304..323
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        304..322
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        604..852
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     604..624
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     775..794
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        873..948
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        961..1340
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1341..1544
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1495..1515
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          1636..1768
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1773..1782
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1779..1781,1782..1784,1785..2498)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1779..1781,1782..1784,1785..2495)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1824..1845)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2085..2104)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2432..2453
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2505..2553
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(2889..2909)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2889..2907)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2928..2944)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2928..2944)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2941..2963)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2952..2968
                     /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(2976..2982,2983..3000,3001..3006))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3021..3042
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3159..3176)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3330..3918)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3410..3429)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4089..4880,4881..4949))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAAKLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4712..4731
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4950..5054)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(5081..5399,1..137)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5168..5187)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5378..5399
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
       61 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
      121 attaacgctt acaatttcca ttcgccattc aggctgcgca actgttggga agggcgatcg
      181 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
      241 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc
      301 gcgtaatacg actcactata gggcgaattg gagctagcgt ttaaacctac agttgaagtc
      361 ggaagtttac atacacttaa gttggagtca ttaaaactcg tttttcaact actccacaaa
      421 tttcttgtta acaaacaata gttttggcaa gtcagttagg acatctactt tgtgcatgac
      481 acaagtcatt tttccaacaa ttgtttacag acagattatt tcacttataa ttcactgtat
      541 cacaattcca gtgggtcaga agtgtacata cacgcgcttg actgtgcctt taagcttggt
      601 accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
      661 gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
      721 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
      781 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
      841 ggacgaaaca ccgggaaacg cggggtacgc tagttttaga gctagaaata gcaagttaaa
      901 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttaagctt
      961 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     1021 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     1081 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     1141 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     1201 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     1261 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
     1321 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
     1381 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     1441 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
     1501 tgggcggtag gcgtgtacgg tgggaggtct atataagcag aggtcgttta gtgaaccgtc
     1561 agatcactag tagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg
     1621 ctcgactgat cacaggtaag tatcaaggtt acaagacagg tttaaggagg ccaatagaaa
     1681 ctgggcttgt cgagacagag aagattcttg cgtttctgat aggcacctat tggtcttact
     1741 gacatccact ttgcctttct ctccacaggg cggccgccat ggtgagcaag ggcgaggagc
     1801 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1861 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     1921 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     1981 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2041 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     2101 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2161 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     2221 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     2281 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     2341 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     2401 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2461 ccgggatcac tctcggcatg gacgagctgt acaagtaaga attcaataaa atatctttat
     2521 tttcattaca tctgtgtgtt ggttttttgt gtgtaattcg agcatcttac cgccatttat
     2581 tcccagatct ttaaacaatt taaaggcaat gctaccaaat actaagcgcg tgtatgtaca
     2641 cttctgaccc actgggaatg tgatgaaaga aataaaagct gaaatgaatc attctctcta
     2701 ctattattct gatatttcac attcttaaaa taaagtggtg atcctaactg accttaagac
     2761 agggaatctt tactcggatt aaatgtcagg aattgtgaaa aagtgagttt aaatgtattt
     2821 ggctaaggtg tatgtaaact tccgacttca actgtagttt aaacgggccc ggtacccagc
     2881 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     2941 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3001 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3061 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3121 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3181 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3241 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3301 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3361 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3421 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3481 tacctgtccg cctttctccc ttcgggaacc gtggcgcttt ctcatagctc acgctgtagg
     3541 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3601 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3661 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3721 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3781 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3841 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3901 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3961 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4021 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4081 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4141 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4201 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4261 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4321 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4381 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4441 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4501 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaactt ggccgcagtg
     4561 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4621 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4681 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4741 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4801 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4861 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4921 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4981 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5041 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg tagcggcgca
     5101 ttaagcgcgg cgggagtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     5161 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5221 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5281 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5341 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgg
//
