LOCUS       sequence-478287-        5581 bp    DNA     circular SYN 11-JUL-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 5581)
  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 5581)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 11, 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: AAV-U6-sgPlxnb2-CRISPRa-3-EF1a-LibVec
FEATURES             Location/Qualifiers
     source          1..5581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        144..392
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     144..164
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     315..334
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        413..488
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        578..1756
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          809..1747
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1704..1724
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             join(1770..1772,1773..1775,1776..2489)
                     /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(1770..1772,1773..1775,1776..2486)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1815..1836)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2076..2095)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2423..2444
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2557..2575)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2558..2575)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2595..2612)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2601..2825
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2844..2984
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2844..2973
                     /label=AAV2 ITR
     rep_origin      3059..3514
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3146..3165)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3356..3377
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3531..3550)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3650..3672
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3710..3728)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3796..3900
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3901..3969,3970..4761)
                     /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     complement(4119..4138)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4932..5520
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5421..5440
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcccccg ttagagggcc tatttcccat gattccttca tatttgcata
      181 tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat
      241 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
      301 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
      361 ctttatatat cttgtggaaa ggacgaaaca ccgggaaacg cggggtacgc tagttttaga
      421 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
      481 gtcggtgctt tttttaatgc aaagatggat aaagttttaa acagagagga atctttgcag
      541 ctaatggacc ttctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc
      601 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg
      661 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
      721 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
      781 tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg
      841 cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacctggc tgcagtacgt
      901 gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta
      961 aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt
     1021 gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta
     1081 aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg
     1141 ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc
     1201 gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg
     1261 ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc
     1321 cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc
     1381 gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc
     1441 gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga
     1501 ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac
     1561 gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt
     1621 ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt
     1681 tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc
     1741 atttcaggtg tcgtgagcgg ccgctcgaga tggtgagcaa gggcgaggag ctgttcaccg
     1801 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1861 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1921 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     1981 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     2041 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     2101 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     2161 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     2221 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     2281 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2341 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     2401 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2461 ctctcggcat ggacgagctg tacaagtaag agcgcacccg tccgagctaa ggcgcgccat
     2521 acatgcatgg tgtacaagta atctagaggg ccctattcta tagtgtcacc taaatgctag
     2581 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     2641 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     2701 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     2761 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     2821 tatggcttct gaggcggggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     2881 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     2941 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     3001 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     3061 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3121 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3181 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3241 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     3301 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3361 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa
     3421 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     3481 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     3541 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     3601 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccttctc cgggagctgc
     3661 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga tacgaaaggg cctcgtgata
     3721 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     3781 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3841 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3901 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3961 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4021 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     4081 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     4141 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     4201 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     4261 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     4321 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     4381 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     4441 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     4501 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     4561 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4621 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4681 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4741 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4801 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4861 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4921 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4981 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5041 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     5101 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5161 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5221 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5281 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5341 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5401 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5461 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5521 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5581 t
//
