LOCUS       sequence-247046-        8637 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Encodes for CRY2-HA and mCherry proteins expressed from the EF1a 
            promoter; 2 gRNAs targeting the Zfp462 promoter and 2 gRNAs 
            targeting the Klf4 SE expressed from their individual hU6 promoters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8637)
  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 8637)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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 + Target  Zfp462-Klf4SE
FEATURES             Location/Qualifiers
     source          1..8637
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             976..996
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1027..1053
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1075..1128
                     /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             1129..1836
                     /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(1271..1289)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1861..1878)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1867..2074
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2083..2223
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2083..2212
                     /label=AAV2 ITR
     rep_origin      2298..2753
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2385..2404)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2595..2616
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2770..2789)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2889..2911
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2949..2967)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3035..3139
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3140..3208,3209..4000)
                     /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(3358..3377)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4171..4759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4660..4679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4821..5069
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        5091..5166
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        5264..5512
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        5534..5609
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        5707..5955
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        5977..6052
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6150..6398
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        6420..6495
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6878..7089
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          7090..8028
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7985..8005
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             8065..8112
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
ORIGIN
        1 attactgctg cggcagaggc gatctgggct tgcagcatcg aagaacttgg tctggaaaac
       61 gaagcggaaa aacccagtaa cgcgctcctg acgcgagcgt ggtcgccagg ctggtcgaat
      121 gctgataagc tccttaatga gtttatcgag aaacagttga tcgactatgc gaagaatagc
      181 aagaaggtgg tgggaaattc cacttccctc ctgtcgccgt atctccactt tggggagatt
      241 agcgtgaggc atgtgttcca atgtgcgcgg atgaagcaaa tcatttgggc tcgcgacaaa
      301 aactctgaag gagaggagtc tgccgatttg tttttgagag gaatcggcct tagggagtat
      361 tcacgataca tctgcttcaa ctttccgttt acacatgaac aatccctcct cagccacctc
      421 cgcttctttc cgtgggacgc ggacgtggac aaattcaagg cctggcggca ggggcgcacg
      481 ggatacccgt tggtggatgc gggtatgaga gagctgtggg caacgggttg gatgcacaac
      541 cgtattcgag tcatcgttag tagctttgcc gttaagttcc tcttgttgcc ctggaaatgg
      601 gggatgaaat acttttggga cacgcttctg gatgcggact tggagtgcga tattcttggg
      661 tggcagtaca tttcgggttc catccctgac gggcatgaac ttgaccgatt ggataaccct
      721 gcactgcaag gagcaaagta cgacccggag ggagaataca tcaggcagtg gctcccagag
      781 ctggcccgtc tgcccaccga gtggattcac cacccgtggg atgctcccct gaccgtcctt
      841 aaagcctcag gggtcgaatt ggggacgaat tacgcgaaac ccatcgtcga tattgacacc
      901 gcacgagaat tgctggcgaa agccatctca agaacacgcg aagcgcagat tatgatcgga
      961 gcagcgccgg ctagccccaa gaagaagaga aaggtggagg ccagcggttc cggacgggct
     1021 gtatactacc cgtacgacgt tccggactac gctctgttta aactgggcag tggagagggc
     1081 agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccagt gagcaagggc
     1141 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     1201 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     1261 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     1321 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     1381 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     1441 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     1501 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaaaacc
     1561 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     1621 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     1681 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     1741 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     1801 cactccaccg gcggcatgga cgagctgtac aagtaagaat tcctagagct cgctgatcag
     1861 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     1921 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     1981 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2041 aggattggga agagaatagc aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg
     2101 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     2161 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     2221 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     2281 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2341 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     2401 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2461 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     2521 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2581 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     2641 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     2701 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     2761 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     2821 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     2881 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     2941 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     3001 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3061 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3121 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     3181 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3241 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3301 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     3361 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     3421 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3481 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3541 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3601 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3661 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3721 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3781 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3841 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3901 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     3961 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4021 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4081 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4141 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4201 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4261 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4321 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4381 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4441 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4501 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4561 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4621 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4681 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4741 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4801 ctggcctttt gctcacatgt gagggcctat ttcccatgat tccttcatat ttgcatatac
     4861 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     4921 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     4981 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     5041 tatatatctt gtggaaagga cgaaacaccg tacatgcagt agtactaagt gttttagagc
     5101 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
     5161 cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta
     5221 gcgcgtgcgc caattctgca gacaaatggc tctagaggca tgtgagggcc tatttcccat
     5281 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
     5341 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     5401 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     5461 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgtttgtgt
     5521 tttagtgtag attgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     5581 aacttgaaaa agtggcaccg agtcggtgct tttttgtttt agagctagaa atagcaagtt
     5641 aaaataaggc tagtccgttt ttagcgcgtg cgccaattct gcagacaaat ggctctagag
     5701 gcatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
     5761 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
     5821 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
     5881 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
     5941 aaaggacgaa acaccgtaaa gaaaagtgtt tatcgagttt tagagctaga aatagcaagt
     6001 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt
     6061 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat
     6121 tctgcagaca aatggctcta gaggcatgtg agggcctatt tcccatgatt ccttcatatt
     6181 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     6241 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     6301 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     6361 tcttggcttt atatatcttg tggaaaggtc gaaacaccga agtgtttatc gagggaaagg
     6421 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     6481 gcaccgagtc ggtgcttttt tgttttagag ctagaaatag caagttaaaa taaggctagt
     6541 ccgtttttag cgcgtgcgcc aattctgcag acaaatggct ctagaggtac ccgttacata
     6601 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     6661 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     6721 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     6781 cggtaaatgg cccgcctggc attgtgccca gtacatgacc ttatgggact ttcctacttg
     6841 gcagtacatc tacgtaaata ctagtgtgcc cgtcagtggg cagagcgcac atcgcccaca
     6901 gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc
     6961 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     7021 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     7081 agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc
     7141 ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt
     7201 cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt
     7261 gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt
     7321 cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct
     7381 gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt
     7441 atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg
     7501 gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc
     7561 cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg
     7621 ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca
     7681 gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa
     7741 aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg
     7801 ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt aggttggggg
     7861 gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca
     7921 gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc
     7981 attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgaaag
     8041 cttggtaccg gatccgccac catgaaaagg ccggcggcca cgaaaaaggc cggccaggca
     8101 aaaaagaaaa agaagatgga caaaaagacg attgtgtggt ttagacgtga ccttcgcatt
     8161 gaggacaacc ctgcgttggc agcggcagcg catgaaggtt cggtattccc tgtctttatc
     8221 tggtgtcccg aagaggaggg gcaattctat cccggtcgcg cgtcccgatg gtggatgaaa
     8281 cagtcgctcg cacatcttag ccagtcgttg aaggcgctcg gctctgatct gacgctcatc
     8341 aaaacacaca atacaatttc tgccattctg gattgcatta gggtgactgg tgccaccaaa
     8401 gttgtgttca atcaccttta tgatcccgta agcctggtca gggatcatac agtaaaagag
     8461 aaacttgtag agaggggaat cagcgtccaa tcgtacaatg gggacttgtt gtatgaaccg
     8521 tgggaaatct attgtgagaa aggcaaacct ttcacttcct tcaattcgta ctggaagaaa
     8581 tgcctcgaca tgtccatcga gtcagtcatg ctgccgccac cttggagact tatgccc
//
