LOCUS       sequence-247041-        5060 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes for 4 gRNAs expressed from their individual hU6 promoters. 
            Two gRNAs target the Zfp462 promoter while the other two target the 
            Klf4 stretch enhancer..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5060)
  AUTHORS   Kim JH, Rege M, Valeri J, Dunagin MC, Metzger A, Titus KR, Gilgenast
            TG, Gong W, Beagan JA, Raj A, Phillips-Cremins JE
  TITLE     LADL: light-activated dynamic looping for endogenous gene expression
            control.
  JOURNAL   Nat Methods. 2019 Jun 24. pii: 10.1038/s41592-019-0436-5. doi: 
            10.1038/s41592-019-0436-5.
  PUBMED    31235883
REFERENCE   2  (bases 1 to 5060)
  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 
            Methods. 2019 Jun 24. pii: 10.1038/s41592-019-0436-5. doi: 
            10.1038/s41592-019-0436-5."
COMMENT     Sequence Label: Empty Bridge Control Zfp462-Klf4SE
FEATURES             Location/Qualifiers
     source          1..5060
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(340..357)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    346..553
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   562..702
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   562..691
                     /label=AAV2 ITR
     rep_origin      777..1232
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(864..883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1074..1095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1249..1268)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1368..1390
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1428..1446)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1514..1618
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1619..1687,1688..2479)
                     /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(1837..1856)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2650..3238
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3139..3158
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3300..3548
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        3570..3645
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3743..3991
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        4013..4088
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4186..4434
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        4456..4531
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4629..4877
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        4899..4974
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tagaggtacc cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
       61 cccgcccatt gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      121 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      181 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct
      241 tatgggactt tcctacttgg cagtacatct acgtatgtat ccgcatctga tgctaccgtg
      301 gttgagttag cgtcggaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt
      361 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
      421 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
      481 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca
      541 ggcatgctgg ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
      601 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
      661 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
      721 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
      781 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
      841 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
      901 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
      961 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     1021 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1081 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     1141 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1201 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     1261 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     1321 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     1381 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     1441 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     1501 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     1561 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     1621 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     1681 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     1741 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     1801 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     1861 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     1921 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     1981 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     2041 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     2101 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     2161 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     2221 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     2281 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg
     2341 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     2401 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     2461 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     2521 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     2581 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     2641 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     2701 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     2761 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     2821 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     2881 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     2941 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3001 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3061 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3121 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3181 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3241 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtg
     3301 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     3361 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     3421 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     3481 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     3541 gaaacaccgt acatgcagta gtactaagtg ttttagagct agaaatagca agttaaaata
     3601 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag
     3661 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag
     3721 acaaatggct ctagaggcat gtgagggcct atttcccatg attccttcat atttgcatat
     3781 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
     3841 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
     3901 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
     3961 tttatatatc ttgtggaaag gacgaaacac cgtttgtgtt ttagtgtaga ttgttttaga
     4021 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     4081 gtcggtgctt ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt
     4141 tagcgcgtgc gccaattctg cagacaaatg gctctagagg catgtgaggg cctatttccc
     4201 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     4261 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     4321 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     4381 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtaaag
     4441 aaaagtgttt atcgagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     4501 tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt ttagagctag aaatagcaag
     4561 ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa atggctctag
     4621 aggcatgtga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     4681 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     4741 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     4801 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     4861 ggaaaggtcg aaacaccgaa gtgtttatcg agggaaaggt tttagagcta gaaatagcaa
     4921 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     4981 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgtttttagc gcgtgcgcca
     5041 attctgcaga caaatggctc
//
