LOCUS       sequence-475925-        7260 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Sleeping-beauty based EV 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 7260)
  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 7260)
  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: pT4-U6-sgRNA-CMV-EGFP
FEATURES             Location/Qualifiers
     source          1..7260
                     /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        2734..2809
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2822..3201
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3202..3405
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3356..3376
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          3497..3629
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      3634..3643
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3640..3642,3643..3645,3646..4359)
                     /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(3640..3642,3643..3645,3646..4356)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3685..3706)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3946..3965)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4293..4314
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4366..4414
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4750..4770)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4750..4768)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4789..4805)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4789..4805)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4802..4824)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4813..4829
                     /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(4837..4843,4844..4861,4862..4867))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4882..4903
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5020..5037)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5191..5779)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5271..5290)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5950..6741,6742..6810))
                     /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     6573..6592
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6811..6915)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6942..7260,1..137)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7029..7048)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7239..7260
                     /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 ccggagacgg ttgtaaatga gcacacaaaa tacacatgct aaaatattat
      901 attctatgac ctttataaaa tcaaccaaaa tcttcttttt aataacttta gtatcaataa
      961 ttagaatttt tatgttcctt tttgcaaact tttaataaaa atgagcaaaa taaaaaaacg
     1021 ctagttttag taactcgcgt tgttttcttc acctttaata atagctactc caccacttgt
     1081 tcctaagcgg tcagctcctg cttcaatcat tttttgagca tcttcaaatg ttctaactcc
     1141 accagctgct ttaactaaag cattgtcttt aacaactgac ttcattagtt taacatcttc
     1201 aaatgttgca cctgattttg aaaatcctgt tgatgtttta acaaattcta atccagcttc
     1261 aacagctatt tcacaagctt tcatgatttc ttcttttgtt aataaacaat tttccataat
     1321 acatttaaca acatgtgatc cagctgcttt ttttacagct ttcatgtctt ctaaaactaa
     1381 ttcataattt ttgtctttta atgcaccaat atttaatacc atatcaattt ctgttgcacc
     1441 atctttaatt gcttcagaaa cttcgaatgc ttttgtagct gttgtgcatg cacctagagg
     1501 aaaacctaca acatttgtta ttcctacatt tgtgcctttt aataattctt tacaatagct
     1561 tgttcaatat gaattaacac aaactgttgc aaaatcaaat tcaattgctt catcacataa
     1621 ttgtttaatt tcagctttcg tagcatcttg ttttaataat gtgtgatcta tatatttgtt
     1681 tagtttcatt ttttctccta tatattcatt tttaatttta attctttaat aatttcgtct
     1741 actttaactt tagcgttttg aacagattca ccaacaccta taaaataaat ttttagttta
     1801 ggttcagttc cacttgggcg aacagcaaat catgacttat cttctaaata aaattttagt
     1861 aagtcttgtc ctggcatatt atacattcca tcgatgtagt cttcaacatt aacaacttta
     1921 agtccagcaa tttgagttaa gggtgttgct ctcaatgatt tcattaatgg ttcaattttt
     1981 aatttctttt cttctggttt aaaattcaag tttaaagtga aagtgtaata tgcacccatt
     2041 tctttaaata aatcttctaa atagtctact aatgttttat tttgtttttt ataaaatcaa
     2101 gcagcctctg ctattaatat agaagcttgt attccatctt tatctctagc tgagtcatca
     2161 attacatatc cataactttc ttcataagca aaaacaaaat ttaatccgtt atcttcttct
     2221 ttagcaattt ctctacccat tcatttaaat ccagttaaag tttttacaat attaactcca
     2281 tatttttcat gagcgattct atcacccaaa tcacttgtta caaaacttga atatagagcc
     2341 ggattttttg gaatgctatt taagcgtttt agatttgata attttcaatc aattaaaatt
     2401 ggtcctgttt gatttccatc taatcttaca aaatgaccat catgttttat tgccattcca
     2461 aatctgtcag catctgggtc attcataata ataatatctg catcatgttt aataccatat
     2521 tcaagcggta tttttcatgc aggatcaaat tctggatttg gatttacaac atttttaaat
     2581 gtttcatctt caaatgcatg ctcttcaacc tcaataacgt tatatcctga ttcacgtaat
     2641 atttttgggg taaatttagt tcctgttcca ttaactgcgc taaaaataat ttttaaatct
     2701 tttttagctt cttgctcttt tttgtacgtc tctgttttag agctagaaat agcaagttaa
     2761 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttaagct
     2821 tggcattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
     2881 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     2941 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     3001 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     3061 caagtgtatc atatgccaag tccgccccct attgacgtca atgacggtaa atggcccgcc
     3121 tggcattatg cccagtacat gaccttacgg gactttccta cttggcagta catctacgta
     3181 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag
     3241 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     3301 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa
     3361 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gaggtcgttt agtgaaccgt
     3421 cagatcacta gtagctttat tgcggtagtt tatcacagtt aaattgctaa cgcagtcagt
     3481 gctcgactga tcacaggtaa gtatcaaggt tacaagacag gtttaaggag gccaatagaa
     3541 actgggcttg tcgagacaga gaagattctt gcgtttctga taggcaccta ttggtcttac
     3601 tgacatccac tttgcctttc tctccacagg gcggccgcca tggtgagcaa gggcgaggag
     3661 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     3721 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     3781 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     3841 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     3901 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     3961 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     4021 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     4081 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     4141 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     4201 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     4261 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     4321 gccgggatca ctctcggcat ggacgagctg tacaagtaag aattcaataa aatatcttta
     4381 ttttcattac atctgtgtgt tggttttttg tgtgtaattc gagcatctta ccgccattta
     4441 ttcccagatc tttaaacaat ttaaaggcaa tgctaccaaa tactaagcgc gtgtatgtac
     4501 acttctgacc cactgggaat gtgatgaaag aaataaaagc tgaaatgaat cattctctct
     4561 actattattc tgatatttca cattcttaaa ataaagtggt gatcctaact gaccttaaga
     4621 cagggaatct ttactcggat taaatgtcag gaattgtgaa aaagtgagtt taaatgtatt
     4681 tggctaaggt gtatgtaaac ttccgacttc aactgtagtt taaacgggcc cggtacccag
     4741 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt
     4801 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4861 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4921 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4981 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5041 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5101 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5161 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5221 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5281 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5341 atacctgtcc gcctttctcc cttcgggaac cgtggcgctt tctcatagct cacgctgtag
     5401 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5461 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5521 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5581 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5641 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5701 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5761 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5821 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5881 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5941 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6001 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6061 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6121 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6181 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6241 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6301 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6361 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaact tggccgcagt
     6421 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6481 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6541 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6601 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6661 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6721 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6781 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6841 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6901 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc
     6961 attaagcgcg gcgggagtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     7021 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     7081 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     7141 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     7201 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
//
