LOCUS       sequence-251077-        3823 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCR template for dual guide RNA cloning, guide RNA scaffold for the 
            Streptococcus pyogenes CRISPR/Cas9 system - H1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3823)
  AUTHORS   Miguel-Escalada I, Bonas-Guarch S, Cebola I, Ponsa-Cobas J, 
            Mendieta-Esteban J, Atla G, Javierre BM, Rolando DMY, Farabella I, 
            Morgan CC, Garcia-Hurtado J, Beucher A, Moran I, Pasquali L, 
            Ramos-Rodriguez M, Appel EVR, Linneberg A, Gjesing AP, Witte DR, 
            Pedersen O, Grarup N, Ravassard P, Torrents D, Mercader JM, Piemonti
            L, Berney T, de Koning EJP, Kerr-Conte J, Pattou F, Fedko IO, Groop 
            L, Prokopenko I, Hansen T, Marti-Renom MA, Fraser P, Ferrer J
  TITLE     Human pancreatic islet three-dimensional chromatin architecture 
            provides insights into the genetics of type 2 diabetes.
  JOURNAL   Nat Genet. 2019 Jun 28. pii: 10.1038/s41588-019-0457-0. doi: 
            10.1038/s41588-019-0457-0.
  PUBMED    31253982
REFERENCE   2  (bases 1 to 3823)
  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 Genet. 
            2019 Jun 28. pii: 10.1038/s41588-019-0457-0. doi: 
            10.1038/s41588-019-0457-0."
COMMENT     Sequence Label: pScaffold-H1 donor
FEATURES             Location/Qualifiers
     source          1..3823
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(345..559)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(405..424)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        complement(566..641)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(711..730)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(712..730)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(737..754)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(737..753)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(746..768)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             891..1193
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1072..1091
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1542..2336
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             1542..2336
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1596..1615)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2206..2225
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2536..2545
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2542..2916
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      3054..3642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3543..3562
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3796..3813
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gcccttggga aagagtggtc tcatacagaa
      361 cttataagac tcccaaatcc aaagacattt cacgtttatg gtgatttccc agaacacata
      421 gcgacatgca aatattgcag ggcgccactc ccctgtccct cacagccatc ttcctgccag
      481 ggcgcacgcg cgctgggtgt tcccgcctag tgacactggg cccgcgattc cttggagcgg
      541 gttgatgacg tcagcgttca aaaaagcacc gactcggtgc cactttttca agttgataac
      601 ggactagcct tattttaact tgctatttct agctctaaaa caagggcgaa ttctgcagat
      661 atccatcaca ctggcggccg ctcgagcatg catctagagg gcccaattcg ccctatagtg
      721 agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
      781 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
      841 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct atacgtacgg cagtttaagg
      901 tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta
      961 ttgacacgcc ggggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata
     1021 aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga
     1081 ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc
     1141 accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag
     1201 gcatgagatt atcaaaaagg atcttcacct agatcctttt cacgtagaaa gccagtccgc
     1261 agaaacggtg ctgaccccgg atgaatgtca gctactgggc tatctggaca agggaaaacg
     1321 caagcgcaaa gagaaagcag gtagcttgca gtgggcttac atggcgatag ctagactggg
     1381 cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg
     1441 ggaagccctg caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg
     1501 gatcaagctc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     1561 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     1621 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     1681 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc
     1741 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     1801 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     1861 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     1921 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     1981 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     2041 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga
     2101 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     2161 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     2221 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     2281 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgaatta
     2341 ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2401 accgcatcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2461 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2521 tagcacgtga ggagggccac catggccaag ttgaccagtg ccgttccggt gctcaccgcg
     2581 cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc
     2641 gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc
     2701 caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag
     2761 ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc
     2821 atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc
     2881 aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctaaaact tcatttttaa
     2941 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3001 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3061 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3121 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3181 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     3241 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3301 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3361 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3421 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3481 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3541 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3601 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3661 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3721 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3781 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aag
//
