LOCUS       sequence-316786-        3970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA targeting human PAX7 3' UTR with 17bp spacer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3970)
  AUTHORS   Xi H, Young CS, Pyle AD
  TITLE     Generation of PAX7 Reporter Cells to Investigate Skeletal Myogenesis
            from Human Pluripotent Stem Cells.
  JOURNAL   STAR Protoc. 2020 Nov 5;1(3):100158. doi: 
            10.1016/j.xpro.2020.100158. eCollection 2020 Dec 18.
  PUBMED    33377052
REFERENCE   2  (bases 1 to 3970)
  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: "STAR 
            Protoc."; date: "2020-11-5"; volume: "1(3):100158. doi"; pages: " 
            10.1016/j.xpro.2020.100158. eCollection 2020 Dec 18"
COMMENT     Sequence Label: pHX_gRNA_hPAX7_6-9S
FEATURES             Location/Qualifiers
     source          1..3970
                     /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"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        267..342
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(354..378)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(463..481)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(464..481)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(499..515)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(499..515)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(512..534)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    523..539
                     /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(547..553,554..571,572..577))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    592..613
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(730..747)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(901..1489)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(981..1000)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1627..2001)
                     /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"
     regulatory      1998..2007
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2207..3001)
                     /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             complement(2207..3001)
                     /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(2318..2337)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2928..2947
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3350..3652)
                     /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     complement(3452..3471)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3775..3797
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3789..3806
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3790..3806
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3813..3832
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3813..3831
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg tgagtgggtg tacgtggttt tagagctaga aatagcaagt taaaataagg
      301 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc tagacccagc
      361 tttcttgtac aaagttggca ttaaagggcg aattccagca cactggcggc cgttactagt
      421 ggatccgagc tcggtaccaa gcttgatgca tagcttgagt attctatagt gtcacctaaa
      481 tagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
      541 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
      601 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
      661 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
      721 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
      781 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
      841 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
      901 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      961 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     1021 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     1081 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     1141 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     1201 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     1261 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     1321 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     1381 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     1441 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     1501 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     1561 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttag
     1621 cacgtgtcag tcctgctcct cggccacgaa gtgcacgcag ttgccggccg ggtcgcgcag
     1681 ggcgaactcc cgcccccacg gctgctcgcc gatctcggtc atggccggcc cggaggcgtc
     1741 ccggaagttc gtggacacga cctccgacca ctcggcgtac agctcgtcca ggccgcgcac
     1801 ccacacccag gccagggtgt tgtccggcac cacctggtcc tggaccgcgc tgatgaacag
     1861 ggtcacgtcg tcccggacca caccggcgaa gtcgtcctcc acgaagtccc gggagaaccc
     1921 gagccggtcg gtccagaact cgaccgctcc ggcgacgtcg cgcgcggtga gcaccggaac
     1981 ggcactggtc aacttggcca tggtggccct cctcacgtgc tattattgaa gcatttatca
     2041 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     2101 ggttccgcgc acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata ccgcacagat
     2161 gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt aataattcag aagaactcgt
     2221 caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga
     2281 ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta
     2341 tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca gaaaagcggc
     2401 cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc
     2461 cgtcgggcat gctcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct
     2521 cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga
     2581 tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc
     2641 gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat gacaggagat
     2701 cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga
     2761 gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtctt
     2821 gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg
     2881 ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc
     2941 cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca
     3001 tgcgaaacga tcctcatcct gtctcttgat cagagcttga tcccctgcgc catcagatcc
     3061 ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg
     3121 ccccagctgg caattccggt tcgcttgctg tccataaaac cgcccagtct agctatcgcc
     3181 atgtaagccc actgcaagct acctgctttc tctttgcgct tgcgttttcc cttgtccaga
     3241 tagcccagta gctgacattc atccggggtc agcaccgttt ctgcggactg gctttctacg
     3301 tgaaaaggat ctaggtgaag atcctttttg ataatctcat gcctgacatt tatattcccc
     3361 agaacatcag gttaatggcg tttttgatgt cattttcgcg gtggctgaga tcagccactt
     3421 cttccccgat aacggagacc ggcacactgg ccatatcggt ggtcatcatg cgccagcttt
     3481 catccccgat atgcaccacc gggtaaagtt cacgggagac tttatctgac agcagacgtg
     3541 cactggccag ggggatcacc atccgtcgcc ccggcgtgtc aataatatca ctctgtacat
     3601 ccacaaacag acgataacgg ctctctcttt tataggtgta aaccttaaac tgccgtacgt
     3661 ataggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     3721 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     3781 cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg
     3841 gccctctaga tgcatgctcg agcggccgcc agtgtgatgg atatctgcag aattcgccct
     3901 ttgtacaaaa aagcaggctt taaaggaacc aattcagtcg actggatccg gtaccaaggt
     3961 cgggcaggaa
//
