LOCUS       sequence-242170-        3226 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For CRISPR-mediated epigenome editing of human BLK gene locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3226)
  AUTHORS   Saint Just Ribeiro M, Tripathi P, Namjou B, Harley JB, Chepelev I
  TITLE     Haplotype-specific chromatin looping reveals genetic interactions of
            regulatory regions modulating gene expression in 8p23.1.
  JOURNAL   Front Genet. 2022 Sep 7;13:1008582. doi: 10.3389/fgene.2022.1008582.
            eCollection 2022.
  PUBMED    36160011
REFERENCE   2  (bases 1 to 3226)
  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: "Front 
            Genet."; date: "2022-09-7"; volume: "13:1008582. doi"; pages: " 
            10.3389/fgene.2022.1008582. eCollection 2022"
COMMENT     Sequence Label: pSPgRNA_BLK_E2-4
FEATURES             Location/Qualifiers
     source          1..3226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(380..400)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(380..398)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(419..435)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(419..435)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(432..454)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    443..459
                     /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)."
     promoter        complement(join(467..473,474..491,492..497))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    512..533
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(650..667)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(821..1409)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(901..920)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1580..2371,2372..2440))
                     /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     2203..2222
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2441..2545)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2571..3026)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2708..2729)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2920..2939
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3153..3175
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3167..3184
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3168..3184
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3194..3213
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3194..3212
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttatcg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gtgcatgtgt gtctagtcgg gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttccgcggt
      361 ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt
      421 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
      481 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
      541 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
      601 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
      661 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
      721 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
      781 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
      841 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
      901 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
      961 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     1021 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     1081 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     1141 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     1201 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     1261 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     1321 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     1381 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     1441 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     1501 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     1561 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     1621 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     1681 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     1741 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     1801 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     1861 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     1921 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     1981 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     2041 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     2101 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     2161 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     2221 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     2281 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     2341 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     2401 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     2461 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     2521 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     2581 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     2641 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     2701 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     2761 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     2821 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     2881 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     2941 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     3001 gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga
     3061 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     3121 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     3181 cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtacc
//
