LOCUS       sequence-249167-        8631 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes for CRY2-HA and mCherry proteins expressed from the EF1a 
            promoter as well as 2 gRNAs targeting the Zfp462 promoter expressed 
            from their individual hU6 promoters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8631)
  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 8631)
  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: LADL Bridge + Promoter Only Target
FEATURES             Location/Qualifiers
     source          1..8631
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      860..1448
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1349..1368
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1510..1758
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1780..1855
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1953..2201
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2223..2298
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2396..2644
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2663..2738
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2836..3084
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        3103..3178
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3561..3772
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          3773..4711
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4668..4688
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4748..4795
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6296..6316
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6347..6373
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6395..6448
                     /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             6449..7156
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(6591..6609)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(7181..7198)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7187..7394
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7403..7543
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7403..7532
                     /label=AAV2 ITR
     rep_origin      7618..8073
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7705..7724)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7915..7936
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8090..8109)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8209..8231
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8269..8287)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8355..8459
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8460..8528,8529..8631,1..689)
                     /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"
ORIGIN
        1 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
       61 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      121 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      181 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      241 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      301 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      361 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      421 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      481 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      541 gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      601 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      661 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
      721 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
      781 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
      841 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
      901 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
      961 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1021 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1081 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1141 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1201 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1261 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1321 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1381 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1441 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1501 ctcacatgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     1561 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     1621 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     1681 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     1741 tggaaaggac gaaacaccgt aaagaaaagt gtttatcgag ttttagagct agaaatagca
     1801 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     1861 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc
     1921 aattctgcag acaaatggct ctagaggcat gtgagggcct atttcccatg attccttcat
     1981 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     2041 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     2101 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     2161 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgaagtgttt atcgagggaa
     2221 aggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     2281 gtggcaccga gtcggtgctt ttttgtttta gagctagaaa tagcaagtta aaataaggct
     2341 agtccgtttt tagcgcgtgc gccaattctg cagacaaatg gctctagagg catgtgaggg
     2401 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     2461 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     2521 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
     2581 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
     2641 caccgggtct tcgagaagac ctgttttaga gctagaaata gcaagttaaa ataaggctag
     2701 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgtttta gagctagaaa
     2761 tagcaagtta aaataaggct agtccgtttt tagcgcgtgc gccaattctg cagacaaatg
     2821 gctctagagg catgtgaggg cctatttccc atgattcctt catatttgca tatacgatac
     2881 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
     2941 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     3001 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     3061 atcttgtgga aaggacgaaa caccgggtct tcgagaagac ctgttttaga gctagaaata
     3121 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     3181 ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt tagcgcgtgc
     3241 gccaattctg cagacaaatg gctctagagg tacccgttac ataacttacg gtaaatggcc
     3301 cgcctggctg accgcccaac gacccccgcc cattgacgtc aatagtaacg ccaataggga
     3361 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     3421 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     3481 ggcattgtgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtaa
     3541 atactagtgt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
     3601 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
     3661 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
     3721 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
     3781 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
     3841 tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg
     3901 ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg
     3961 gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc
     4021 tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg
     4081 gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc
     4141 cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg
     4201 agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc
     4261 tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc
     4321 agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag
     4381 gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc
     4441 gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga
     4501 ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat
     4561 ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta
     4621 attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac
     4681 agtggttcaa agtttttttc ttccatttca ggtgtcgtga aagcttggta ccggatccgc
     4741 caccatgaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaagaagat
     4801 ggacaaaaag acgattgtgt ggtttagacg tgaccttcgc attgaggaca accctgcgtt
     4861 ggcagcggca gcgcatgaag gttcggtatt ccctgtcttt atctggtgtc ccgaagagga
     4921 ggggcaattc tatcccggtc gcgcgtcccg atggtggatg aaacagtcgc tcgcacatct
     4981 tagccagtcg ttgaaggcgc tcggctctga tctgacgctc atcaaaacac acaatacaat
     5041 ttctgccatt ctggattgca ttagggtgac tggtgccacc aaagttgtgt tcaatcacct
     5101 ttatgatccc gtaagcctgg tcagggatca tacagtaaaa gagaaacttg tagagagggg
     5161 aatcagcgtc caatcgtaca atggggactt gttgtatgaa ccgtgggaaa tctattgtga
     5221 gaaaggcaaa cctttcactt ccttcaattc gtactggaag aaatgcctcg acatgtccat
     5281 cgagtcagtc atgctgccgc caccttggag acttatgccc attactgctg cggcagaggc
     5341 gatctgggct tgcagcatcg aagaacttgg tctggaaaac gaagcggaaa aacccagtaa
     5401 cgcgctcctg acgcgagcgt ggtcgccagg ctggtcgaat gctgataagc tccttaatga
     5461 gtttatcgag aaacagttga tcgactatgc gaagaatagc aagaaggtgg tgggaaattc
     5521 cacttccctc ctgtcgccgt atctccactt tggggagatt agcgtgaggc atgtgttcca
     5581 atgtgcgcgg atgaagcaaa tcatttgggc tcgcgacaaa aactctgaag gagaggagtc
     5641 tgccgatttg tttttgagag gaatcggcct tagggagtat tcacgataca tctgcttcaa
     5701 ctttccgttt acacatgaac aatccctcct cagccacctc cgcttctttc cgtgggacgc
     5761 ggacgtggac aaattcaagg cctggcggca ggggcgcacg ggatacccgt tggtggatgc
     5821 gggtatgaga gagctgtggg caacgggttg gatgcacaac cgtattcgag tcatcgttag
     5881 tagctttgcc gttaagttcc tcttgttgcc ctggaaatgg gggatgaaat acttttggga
     5941 cacgcttctg gatgcggact tggagtgcga tattcttggg tggcagtaca tttcgggttc
     6001 catccctgac gggcatgaac ttgaccgatt ggataaccct gcactgcaag gagcaaagta
     6061 cgacccggag ggagaataca tcaggcagtg gctcccagag ctggcccgtc tgcccaccga
     6121 gtggattcac cacccgtggg atgctcccct gaccgtcctt aaagcctcag gggtcgaatt
     6181 ggggacgaat tacgcgaaac ccatcgtcga tattgacacc gcacgagaat tgctggcgaa
     6241 agccatctca agaacacgcg aagcgcagat tatgatcgga gcagcgccgg ctagccccaa
     6301 gaagaagaga aaggtggagg ccagcggttc cggacgggct gtatactacc cgtacgacgt
     6361 tccggactac gctctgttta aactgggcag tggagagggc agaggaagtc tgctaacatg
     6421 cggtgacgtc gaggagaatc ctggcccagt gagcaagggc gaggaggata acatggccat
     6481 catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt
     6541 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     6601 ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta
     6661 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     6721 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     6781 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     6841 caacttcccc tccgacggcc ccgtaatgca gaagaaaacc atgggctggg aggcctcctc
     6901 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct
     6961 gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt
     7021 gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga
     7081 ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga
     7141 cgagctgtac aagtaagaat tcctagagct cgctgatcag cctcgactgt gccttctagt
     7201 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     7261 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     7321 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc
     7381 aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     7441 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     7501 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     7561 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     7621 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     7681 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     7741 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     7801 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     7861 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     7921 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     7981 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     8041 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     8101 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     8161 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     8221 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     8281 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     8341 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     8401 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     8461 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     8521 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     8581 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt t
//
