LOCUS       sequence-478292-        5582 bp    DNA     circular SYN 11-JUL-2025
DEFINITION  expresses non-targeting (NT) guide#2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5582)
  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 5582)
  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-sgNT-2-EF1a-LibVec
FEATURES             Location/Qualifiers
     source          1..5582
                     /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        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        579..1757
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          810..1748
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1705..1725
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             join(1771..1773,1774..1776,1777..2490)
                     /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(1771..1773,1774..1776,1777..2487)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1816..1837)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2077..2096)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2424..2445
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2558..2576)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2559..2576)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2596..2613)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2602..2826
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2845..2985
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2845..2974
                     /label=AAV2 ITR
     rep_origin      3060..3515
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3147..3166)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3357..3378
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3532..3551)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3651..3673
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3711..3729)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3797..3901
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3902..3970,3971..4762)
                     /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(4120..4139)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4933..5521
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5422..5441
                     /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 ccgaacctac gggctacgat acggttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct ttttttaatg caaagatgga taaagtttta aacagagagg aatctttgca
      541 gctaatggac cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg
      601 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
      661 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
      721 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
      781 ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg
      841 gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg
      901 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt
      961 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
     1021 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt
     1081 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg
     1141 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
     1201 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac
     1261 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg
     1321 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
     1381 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg
     1441 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg
     1501 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
     1561 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg
     1621 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt
     1681 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
     1741 catttcaggt gtcgtgagcg gccgctcgag atggtgagca agggcgagga gctgttcacc
     1801 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1861 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1921 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1981 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     2041 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     2101 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2161 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     2221 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     2281 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     2341 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     2401 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     2461 actctcggca tggacgagct gtacaagtaa gagcgcaccc gtccgagcta aggcgcgcca
     2521 tacatgcatg gtgtacaagt aatctagagg gccctattct atagtgtcac ctaaatgcta
     2581 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2641 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2701 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2761 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     2821 ctatggcttc tgaggcgggg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     2881 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     2941 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     3001 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     3061 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3121 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3181 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3241 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     3301 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3361 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     3421 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3481 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     3541 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     3601 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccttct ccgggagctg
     3661 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg atacgaaagg gcctcgtgat
     3721 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     3781 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3841 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3901 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3961 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4021 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4081 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4141 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4201 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4261 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4321 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4381 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4441 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4501 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4561 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4621 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4681 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4741 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4801 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4861 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4921 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4981 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5041 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     5101 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5161 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5221 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5281 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5341 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5401 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5461 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5521 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5581 gt
//
