LOCUS       sequence-424547-        4580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  provide ""sgRNA scaffold-spacer-mouse U6"" fragment.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4580)
  AUTHORS   Ren X, Yang H, Nierenberg JL, Sun Y, Chen J, Beaman C, Pham T, 
            Nobuhara M, Takagi MA, Narayan V, Li Y, Ziv E, Shen Y
  TITLE     High-throughput PRIME-editing screens identify functional DNA 
            variants in the human genome.
  JOURNAL   Mol Cell. 2023 Dec 21;83(24):4633-4645.e9. doi: 
            10.1016/j.molcel.2023.11.021.
  PUBMED    38134886
REFERENCE   2  (bases 1 to 4580)
  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: 
            "10.1016/j.molcel.2023.11"; journalName: "Mol Cell"; date: 
            "2023-12-21- 21"; volume: "83"; issue: "24"; pages: "4633-4645.e9"
COMMENT     Sequence Label: sgRNA scaffold-spacer-mouse U6
FEATURES             Location/Qualifiers
     source          1..4580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        159..538
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        539..742
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     693..713
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     739..763
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        949..1262
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     1181..1201
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     intron          1379..1511
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1494..1513
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1556..1574
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1584..2519
                     /codon_start=1
                     /product="luciferase from the anthozoan coelenterate 
                     Renilla reniformis (sea pansy)"
                     /label=Rluc
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     polyA_signal    2550..2671
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2561..2580)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2615..2634
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2804..2908
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2909..2977,2978..3769)
                     /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(3127..3146)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3940..4528
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4429..4448
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcacatggc tcgacagatc ttcaatattg gccattagcc atattattca ttggttatat
       61 agcataaatc aatattggct attggccatt gcatacgttg tatctatatc ataatatgta
      121 catttatatt ggctcatgtc caatatgacc gccatgttgg cattgattat tgactagtta
      181 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      241 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      301 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      361 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtcc
      421 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      481 cttacgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      541 gatgcggttt tggcagtaca ccaatgggcg tggatagcgg tttgactcac ggggatttcc
      601 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      661 tccaaaatgt cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg
      721 ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcactagaa gcttcgtctc
      781 agtttcagag ctatgctgga aacagcatag caagttgaaa taaggctagt ccgttatcaa
      841 cttgaaaaag tggcaccgag tcggtgcttt ttttgtcgag tactaggatc cattaggcgg
      901 ccgcgtggat aaccgtatta ccgccatgca ttagttatta gatctagaga tccgacgccg
      961 ccatctctag gcccgcgccg gccccctcgc acagacttgt gggagaagct cggctactcc
     1021 cctgccccgg ttaatttgca tataatattt cctagtaact atagaggctt aatgtgcgat
     1081 aaaagacaga taatctgttc tttttaatac tagctacatt ttacatgata ggcttggatt
     1141 tctataagag atacaaatac taaattatta ttttaaaaaa cagcacaaaa ggaaactcac
     1201 cctaactgta aagtaattgt gtgttttgag actataaata tcccttggag aaaagccttg
     1261 tttgagacga agctttattg cggtagttta tcacagttaa attgctaacg cagtcagtgc
     1321 ttctgacaca acagtctcga acttaagctg cagaagttgg tcgtgaggca ctgggcaggt
     1381 aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct tgtcgagaca
     1441 gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc actttgcctt
     1501 tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga gtacttaata
     1561 cgactcacta taggctagcc accatgactt cgaaagttta tgatccagaa caaaggaaac
     1621 ggatgataac tggtccgcag tggtgggcca gatgtaaaca aatgaatgtt cttgattcat
     1681 ttattaatta ttatgattca gaaaaacatg cagaaaatgc tgttattttt ttacatggta
     1741 acgcggcctc ttcttattta tggcgacatg ttgtgccaca tattgagcca gtagcgcggt
     1801 gtattatacc agaccttatt ggtatgggca aatcaggcaa atctggtaat ggttcttata
     1861 ggttacttga tcattacaaa tatcttactg catggtttga acttcttaat ttaccaaaga
     1921 agatcatttt tgtcggccat gattggggtg cttgtttggc atttcattat agctatgagc
     1981 atcaagataa gatcaaagca atagttcacg ctgaaagtgt agtagatgtg attgaatcat
     2041 gggatgaatg gcctgatatt gaagaagata ttgcgttgat caaatctgaa gaaggagaaa
     2101 aaatggtttt ggagaataac ttcttcgtgg aaaccatgtt gccatcaaaa atcatgagaa
     2161 agttagaacc agaagaattt gcagcatatc ttgaaccatt caaagagaaa ggtgaagttc
     2221 gtcgtccaac attatcatgg cctcgtgaaa tcccgttagt aaaaggtggt aaacctgacg
     2281 ttgtacaaat tgttaggaat tataatgctt atctacgtgc aagtgatgat ttaccaaaaa
     2341 tgtttattga atcggaccca ggattctttt ccaatgctat tgttgaaggt gccaagaagt
     2401 ttcctaatac tgaatttgtc aaagtaaaag gtcttcattt ttcgcaagaa gatgcacctg
     2461 atgaaatggg aaaatatatc aaatcgttcg ttgagcgagt tctcaaaaat gaacaataat
     2521 tctagagcgg ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac
     2581 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     2641 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     2701 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     2761 taaaatcgat aaggatccag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2821 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     2881 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     2941 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3001 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3061 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3121 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     3181 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     3241 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     3301 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3361 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3421 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     3481 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3541 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     3601 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     3661 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     3721 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     3781 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     3841 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     3901 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3961 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4021 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4081 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4141 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4201 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4261 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4321 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4381 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4441 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4501 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4561 ggccttttgc tggccttttg
//
