LOCUS       sequence-481874-        5499 bp    DNA     circular SYN 08-AUG-2025
DEFINITION  Vector for expressing an sgRNA targeting CLTA promoter from the 
            mouse U6 promoter and a puromycin resistance cassette and BFP from 
            the EF1Alpha promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5499)
  AUTHORS   Xu D, Besselink S, Ramadoss GN, Dierks PH, Lubin JP, Pattali RK, 
            Brim JI, Christenson AE, Colias PJ, Ornelas IJ, Nguyen CD, Chasins 
            SE, Conklin BR, Nunez JK
  TITLE     Programmable epigenome editing by transient delivery of CRISPR 
            epigenome editor ribonucleoproteins.
  JOURNAL   bioRxiv [Preprint]. 2024 Nov 28:2024.11.26.625496. doi: 
            10.1101/2024.11.26.625496.
  PUBMED    39651312
REFERENCE   2  (bases 1 to 5499)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 8, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Nov 28:2024.11.26.625496. doi: 
            10.1101/2024.11.26.625496."
COMMENT     Sequence Label: mU6-sgRNA-CLTA-mCherry
FEATURES             Location/Qualifiers
     source          1..5499
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     977..996
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    995..1050
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     1096..1115
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(1202..1912)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1480..1499)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1749..1767
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      1909..1918
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1925..1978)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(1988..2584)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2068..2088)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2565..2584
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     intron          2605..3543
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2628..2648)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(3544..3755)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     primer_bind     complement(4000..4020)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    4024..4057
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4364..4468
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4469..4537,4538..5329)
                     /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(4687..4706)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt catatgtaga
      661 gaggtacctc gagcggcccg agaagaggga cagctatgac tgggagtagt caggagagga
      721 ggaaaaatct ggctagtaaa acatgtaagg aaaattttag ggatgttaaa gaaaaaaata
      781 acacaaaaca aaatataaaa aaaatctaac ctcaagtcaa ggcttttcta tggaataagg
      841 aatggacagc agggggctgt ttcatatact gatgacctct ttatagccaa cctttgttca
      901 tggcagccag catatgggca tatgttgcca aactctaaac caaatactca ttctgatgtt
      961 ttaaatgatt tgccctccca tatgtccttc cgagtgagag acacaaaaaa ttccaacaca
     1021 ctattgcaat gaaaataaat ttcctttatt agccagaagt cagatgctca aggggcttca
     1081 tgatgtcccc ataatttttg gcagagggaa aaagatctca gtggtatttg tgagccaggg
     1141 cattggccac accagccacc accttctgat aggcagcctg cacctgagga gtgcggccgc
     1201 tttacttgta cagctcgtcc atgccgccgg tggagtggcg gccctcggcg cgttcgtact
     1261 gttccacgat ggtgtagtcc tcgttgtggg aggtgatgtc caacttgatg ttgacgttgt
     1321 aggcgccggg cagctgcacg ggcttcttgg ccttgtaggt ggtcttgacc tcagcgtcgt
     1381 agtggccgcc gtccttcagc ttcagcctct gcttgatctc gcccttcagg gcgccgtcct
     1441 cggggtacat ccgctcggag gaggcctccc agcccatggt cttcttctgc attacggggc
     1501 cgtcggaggg gaagttggtg ccgcgcagct tcaccttgta gatgaactcg ccgtcctgca
     1561 gggaggagtc ctgggtcacg gtcaccacgc cgccgtcctc gaagttcatc acgcgctccc
     1621 acttgaagcc ctcggggaag gacagcttca agtagtcggg gatgtcggcg gggtgcttca
     1681 cgtaggcctt ggagccgtac atgaactgag gggacaggat gtcccaggcg aagggcaggg
     1741 ggccaccctt ggtcaccttc agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc
     1801 cctcgccctc gatctcgaac tcgtggccgt tcacggagcc ctccatgtgc accttgaagc
     1861 gcatgaactc cttgatgatg gccatgttat cctcctcgcc cttgctcacc atggtggcga
     1921 ccggtgggcc aggattctcc tcgacgtcac cgcatgttag cagacttcct ctgccctctc
     1981 ccgatccggc accgggcttg cgggtcatgc accaggtgcg cggtccttcg ggcacctcga
     2041 cgtcggcggt gacggtgaag ccgagccgct cgtagaaggg gaggttgcgg ggcgcggagg
     2101 tctccaggaa ggcgggcacc ccggcgcgct cggccgcctc cactccgggg agcacgacgg
     2161 cgctgcccag acccttgccc tggtggtcgg gcgagacgcc gacggtggcc aggaaccacg
     2221 cgggctcctt gggccggtgc ggcgccagga ggccttccat ctgttgctgc gcggccagcc
     2281 gggaaccgct caactcggcc atgcgcgggc cgatctcggc gaacaccgcc cccgcttcga
     2341 cgctctccgg cgtggtccag accgccaccg cggcgccgtc gtccgcgacc cacaccttgc
     2401 cgatgtcgag cccgacgcgc gtgaggaaga gttcttgcag ctcggtgacc cgctcgatgt
     2461 ggcggtccgg gtcgacggtg tggcgcgtgg cggggtagtc ggcgaacgcg gcggcgaggg
     2521 tgcgtacggc ccgggggacg tcgtcgcggg tggcgaggcg caccgtgggc ttgtactcgg
     2581 tcatggtggg ctagctcacg acacctgaaa tggaagaaaa aaactttgaa ccactgtctg
     2641 aggcttgaga atgaaccaag atccaaactc aaaaagggca aattccaagg agaattacat
     2701 caagtgccaa gctggcctaa cttcagtctc cacccactca gtgtggggaa actccatcgc
     2761 ataaaacccc tccccccaac ctaaagacga cgtactccaa aagcgcgaga actaatcgag
     2821 gtgcctggac ggcgcccggt actccgtgga gtcacatgaa gcgacggctg aggacggaaa
     2881 ggcccttttc ctttgtgtgg gtgactcacc cgcccgctct cccgagcgcc gcgtcctcca
     2941 ttttgagctc cctgcagcag ggccgggaag cggccatctt tccgctcacg caactggtgc
     3001 cgaccgggcc agccttgccg cccagggcgg ggcgatacac ggcggcgcga ggccaggcac
     3061 cagagcaggc cggccagctt gagactaccc ccgtccgatt ctcggtggcc gcgctcgcag
     3121 gccccgcctc gccgaacatg tgcgctggga cgcacgggcc ccgtcgccgc ccgcggcccc
     3181 aaaaaccgaa ataccagtgt gcagatcttg gcccgcattt acaagactat cttgccagaa
     3241 aaaaagcgtc gcagcaggtc atcaaaaatt ttaaatggct agagacttat cgaaagcagc
     3301 gagacaggcg cgaaggtgcc accagattcg cacgcggcgg ccccagcgcc caggccaggc
     3361 ctcaactcaa gcacgaggcg aaggggctcc ttaagcgcaa ggcctcgaac tctcccaccc
     3421 acttccaacc cgaagctcgg gatcaagaat cacgtactgc agccaggtgg aagtaattca
     3481 aggcacgcaa gggccataac ccgtaaagag gccaggcccg cgggaaccac acacggcact
     3541 tacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga ctactgcact
     3601 tatatacggt tctcccccac cctcgggaaa aaggcggagc cagtacacga catcactttc
     3661 ccagtttacc ccgcgccacc ttctctaggc accggttcaa ttgccgaccc ctccccccaa
     3721 cttctcgggg actgtgggcg atgtgcgctc tgcccactga cgggcacatg catggcggta
     3781 atacggttat ccacgcggcc gcctaatgga tcctagtact cgagaaaaaa agcaccgact
     3841 cggtgccact ttttcaagtt gataacggac tagccttatt taaacttgct atgctgtttc
     3901 cagcttagct cttaaacctg tggtgccgac tgggagccaa caaggtggtt ctccaaggga
     3961 tacttatagt ctcaaaacac acaattactt tacagttagg gtgagtttcc ttttgtgctg
     4021 tttataactt cgtataatgt atgctatacg aagttataga aatccaagcc tatcatgtaa
     4081 aatgtagcta gtattaaaaa gaacagatta tctgtctttt atcgcacatt aagcctctat
     4141 agttactagg aaatattata tgcaaattaa ccggggcagg ggagtagccg agcttctccc
     4201 acaagtccgt gcgagggggc cggcgcgggc ctagagatgg cgcgtcggat ctctagattc
     4261 ctgcccgacc ttggtaccgg atccagtcga ctgaattggt tcctttaaag cctgcttttt
     4321 tgtacagcta gcgacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4381 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4441 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4501 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4561 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     4621 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     4681 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     4741 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4801 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     4861 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     4921 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     4981 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     5041 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     5101 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     5161 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     5221 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     5281 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     5341 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5401 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5461 ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
