LOCUS       sequence-350137-        3006 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control vector for coselection for PE3 in human cells. Tandem 
            expression of ATP1A1 G3 and EBFP Nick sgRNAs from two independent U6
            promoters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3006)
  AUTHORS   Levesque S, Mayorga D, Fiset JP, Goupil C, Duringer A, Loiselle A, 
            Bouchard E, Agudelo D, Doyon Y
  TITLE     Marker-free co-selection for successive rounds of prime editing in 
            human cells.
  JOURNAL   Nat Commun. 2022 Oct 7;13(1):5909. doi: 10.1038/s41467-022-33669-z.
  PUBMED    36207338
REFERENCE   2  (bases 1 to 3006)
  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"; journalName: "Nat 
            Commun."; date: "2022-10-7"; volume: "13(1):5909. doi"; pages: " 
            10.1038/s41467-022-33669-z"
COMMENT     Sequence Label: ATP1A1_G3_EBFP_Nick_Dual_sgRNA
FEATURES             Location/Qualifiers
     source          1..3006
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        465..713
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        734..809
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(835..851)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(835..851)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(848..870)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    859..875
                     /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(883..889,890..907,908..913))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    928..949
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1066..1083)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1237..1825)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1317..1336)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1996..2787,2788..2856))
                     /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     2619..2638
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2857..2961)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ctgaagttca tctgcaccac gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc gtcgactgga tccggtacca aggtcgggca ggaagagggc ctatttccca
      481 tgattccttc atatttgcat atacgataca aggctgttag agagataatt agaattaatt
      541 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
      601 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
      661 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgagttct
      721 gtaattcagc atagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
      781 aacttgaaaa agtggcaccg agtcggtgct ttttttaagc ttggcgtaat catggtcata
      841 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
      901 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
      961 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1021 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     1081 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     1141 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     1201 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     1261 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1321 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1381 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     1441 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     1501 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     1561 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     1621 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     1681 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     1741 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     1801 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     1861 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     1921 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     1981 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     2041 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     2101 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     2161 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     2221 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     2281 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     2341 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     2401 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     2461 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     2521 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     2581 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     2641 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     2701 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     2761 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     2821 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     2881 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     2941 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgctagctg
     3001 tacagt
//
