LOCUS       sequence-440825-        7438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express the gRNA targeting the FOXA-binding site in the human ZEB2 
            locus for the CRISPRd experiment.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7438)
  AUTHORS   Matsui S, Granitto M, Buckley M, Ludwig K, Koigi S, Shiley J, 
            Zacharias WJ, Mayhew CN, Lim HW, Iwafuchi M
  TITLE     Pioneer and PRDM transcription factors coordinate bivalent 
            epigenetic states to safeguard cell fate.
  JOURNAL   Mol Cell. 2024 Jan 4:S1097-2765(23)01026-2. doi: 
            10.1016/j.molcel.2023.12.007.
  PUBMED    38211589
REFERENCE   2  (bases 1 to 7438)
  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: "Mol Cell. 
            2024 Jan 4:S1097-2765(23)01026-2. doi: 
            10.1016/j.molcel.2023.12.007."
COMMENT     Sequence Label: LRG-sgRNA.FOXA-ZEB2_CRISPRd_v2
FEATURES             Location/Qualifiers
     source          1..7438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     172..193
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    254..842
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(307..327)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(725..736)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             914..1147
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1225..1346
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1262..1281)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1316..1335
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1386..1521
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1541..1560)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1542..1560)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1570..1587)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1570..1586)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1579..1601)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1728..2183
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1815..1834)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2025..2046
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2209..2313
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2314..2382,2383..3174)
                     /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(2532..2551)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3345..3933
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3834..3853
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4087..4104
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4221..4242
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4257..4262,4263..4280,4281..4287)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4295..4311
                     /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)."
     primer_bind     4300..4322
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4319..4335
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4319..4335
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4354..4374
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4356..4374
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4402..4628
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             4629..4809
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4856..4981
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5474..5707
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5892..5936
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        6114..6362
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6114..6134
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6285..6304
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6384..6459
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    6507..6624
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6693..6904
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6924..6933
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6933..6956
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(6957..6959,6960..6962,6963..7438,1..238)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6957..6959,6960..6962,6963..7438,1..235)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7002..7023)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7263..7282)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
       61 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
      121 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
      181 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtagac
      241 gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
      301 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
      361 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
      421 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
      481 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
      541 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
      601 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
      661 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
      721 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
      781 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
      841 gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
      901 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct
      961 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     1021 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     1081 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     1141 tctagcagta cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     1201 aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa
     1261 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     1321 ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc
     1381 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     1441 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     1501 gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta
     1561 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     1621 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     1681 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     1741 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1801 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     1861 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1921 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     1981 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2041 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2101 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     2161 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     2221 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2281 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2341 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2401 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2461 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2521 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2581 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2641 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2701 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2761 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2821 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     2881 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2941 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3001 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     3061 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3121 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3181 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3241 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3301 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3361 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3421 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3481 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3541 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3601 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3661 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3721 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3781 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3841 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3901 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3961 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4021 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4081 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     4141 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     4201 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     4261 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     4321 ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa
     4381 aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg
     4441 taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg
     4501 tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga
     4561 ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat
     4621 acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     4681 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     4741 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     4801 tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc
     4861 tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt
     4921 gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt
     4981 cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg
     5041 aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc
     5101 agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca
     5161 accatccctt cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct
     5221 ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag acaagataga
     5281 ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg
     5341 gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa
     5401 ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa
     5461 gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg
     5521 gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc
     5581 agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct
     5641 ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac
     5701 agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga
     5761 atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt
     5821 gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa
     5881 accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga
     5941 attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag
     6001 gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg
     6061 gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc agagagggcc
     6121 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta
     6181 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     6241 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     6301 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     6361 ccgcccagta aacatcccct gaggttttag agctagaaat agcaagttaa aataaggcta
     6421 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgaatt ctagatcttg
     6481 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     6541 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     6601 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg
     6661 cgccggctcg agtggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     6721 cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt
     6781 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     6841 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     6901 acaggtgtcg tgacgcggga tccgccacca tggattacaa agacgatgac gataagatgg
     6961 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     7021 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     7081 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     7141 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     7201 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     7261 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     7321 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     7381 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacgg
//
