LOCUS       sequence-478285-        7442 bp    DNA     circular SYN 11-JUL-2025
DEFINITION  AAV vector for cloning and expression of sgRNAs - concomitant 
            expression of GFP serves as transfection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7442)
  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 7442)
  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-sgRNA-EF1a-LibVec
FEATURES             Location/Qualifiers
     source          1..7442
                     /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        2274..2349
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2439..3617
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2670..3608
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3565..3585
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             join(3631..3633,3634..3636,3637..4350)
                     /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(3631..3633,3634..3636,3637..4347)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3676..3697)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3937..3956)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4284..4305
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(4418..4436)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4419..4436)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4456..4473)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4462..4686
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4705..4845
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4705..4834
                     /label=AAV2 ITR
     rep_origin      4920..5375
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5007..5026)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5217..5238
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5392..5411)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5511..5533
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5571..5589)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5657..5761
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5762..5830,5831..6622)
                     /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(5980..5999)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6793..7381
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7282..7301
                     /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 ccggagacgg ttgtaaatga gcacacaaaa
      421 tacacatgct aaaatattat attctatgac ctttataaaa tcaaccaaaa tcttcttttt
      481 aataacttta gtatcaataa ttagaatttt tatgttcctt tttgcaaact tttaataaaa
      541 atgagcaaaa taaaaaaacg ctagttttag taactcgcgt tgttttcttc acctttaata
      601 atagctactc caccacttgt tcctaagcgg tcagctcctg cttcaatcat tttttgagca
      661 tcttcaaatg ttctaactcc accagctgct ttaactaaag cattgtcttt aacaactgac
      721 ttcattagtt taacatcttc aaatgttgca cctgattttg aaaatcctgt tgatgtttta
      781 acaaattcta atccagcttc aacagctatt tcacaagctt tcatgatttc ttcttttgtt
      841 aataaacaat tttccataat acatttaaca acatgtgatc cagctgcttt ttttacagct
      901 ttcatgtctt ctaaaactaa ttcataattt ttgtctttta atgcaccaat atttaatacc
      961 atatcaattt ctgttgcacc atctttaatt gcttcagaaa cttcgaatgc ttttgtagct
     1021 gttgtgcatg cacctagagg aaaacctaca acatttgtta ttcctacatt tgtgcctttt
     1081 aataattctt tacaatagct tgttcaatat gaattaacac aaactgttgc aaaatcaaat
     1141 tcaattgctt catcacataa ttgtttaatt tcagctttcg tagcatcttg ttttaataat
     1201 gtgtgatcta tatatttgtt tagtttcatt ttttctccta tatattcatt tttaatttta
     1261 attctttaat aatttcgtct actttaactt tagcgttttg aacagattca ccaacaccta
     1321 taaaataaat ttttagttta ggttcagttc cacttgggcg aacagcaaat catgacttat
     1381 cttctaaata aaattttagt aagtcttgtc ctggcatatt atacattcca tcgatgtagt
     1441 cttcaacatt aacaacttta agtccagcaa tttgagttaa gggtgttgct ctcaatgatt
     1501 tcattaatgg ttcaattttt aatttctttt cttctggttt aaaattcaag tttaaagtga
     1561 aagtgtaata tgcacccatt tctttaaata aatcttctaa atagtctact aatgttttat
     1621 tttgtttttt ataaaatcaa gcagcctctg ctattaatat agaagcttgt attccatctt
     1681 tatctctagc tgagtcatca attacatatc cataactttc ttcataagca aaaacaaaat
     1741 ttaatccgtt atcttcttct ttagcaattt ctctacccat tcatttaaat ccagttaaag
     1801 tttttacaat attaactcca tatttttcat gagcgattct atcacccaaa tcacttgtta
     1861 caaaacttga atatagagcc ggattttttg gaatgctatt taagcgtttt agatttgata
     1921 attttcaatc aattaaaatt ggtcctgttt gatttccatc taatcttaca aaatgaccat
     1981 catgttttat tgccattcca aatctgtcag catctgggtc attcataata ataatatctg
     2041 catcatgttt aataccatat tcaagcggta tttttcatgc aggatcaaat tctggatttg
     2101 gatttacaac atttttaaat gtttcatctt caaatgcatg ctcttcaacc tcaataacgt
     2161 tatatcctga ttcacgtaat atttttgggg taaatttagt tcctgttcca ttaactgcgc
     2221 taaaaataat ttttaaatct tttttagctt cttgctcttt tttgtacgtc tctgttttag
     2281 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     2341 agtcggtgct ttttttaatg caaagatgga taaagtttta aacagagagg aatctttgca
     2401 gctaatggac cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg
     2461 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
     2521 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
     2581 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
     2641 ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg
     2701 gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg
     2761 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt
     2821 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
     2881 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt
     2941 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg
     3001 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
     3061 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac
     3121 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg
     3181 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
     3241 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg
     3301 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg
     3361 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
     3421 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg
     3481 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt
     3541 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
     3601 catttcaggt gtcgtgagcg gccgctcgag atggtgagca agggcgagga gctgttcacc
     3661 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     3721 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     3781 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     3841 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     3901 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     3961 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     4021 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     4081 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     4141 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     4201 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     4261 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     4321 actctcggca tggacgagct gtacaagtaa gagcgcaccc gtccgagcta aggcgcgcca
     4381 tacatgcatg gtgtacaagt aatctagagg gccctattct atagtgtcac ctaaatgcta
     4441 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     4501 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     4561 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     4621 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     4681 ctatggcttc tgaggcgggg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     4741 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     4801 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     4861 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     4921 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4981 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5041 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5101 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     5161 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     5221 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     5281 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     5341 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     5401 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     5461 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccttct ccgggagctg
     5521 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg atacgaaagg gcctcgtgat
     5581 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     5641 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5701 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5761 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5821 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5881 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5941 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     6001 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6061 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6121 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6181 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6241 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6301 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6361 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6421 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6481 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6541 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6601 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6661 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6721 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6781 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6841 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6901 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6961 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     7021 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7081 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7141 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7201 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7261 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7321 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7381 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7441 gt
//
