LOCUS       sequence-416400-        5103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An empty gRNA expression vector to clone U6 promoter driven sgRNAs 
            with gRNA scaffold and with co-expression of DsRed. sgRNA can be 
            cloned by Golden Gate Assembly or restriction digestion using BbsI..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5103)
  AUTHORS   Rajaram N, Bashtrykov P, Jeltsch A
  TITLE     Protocol for Allele-Specific Epigenome Editing Using CRISPR/dCas9.
  JOURNAL   Methods Mol Biol. 2024;2842:179-192. doi: 
            10.1007/978-1-0716-4051-7_9.
  PUBMED    39012596
REFERENCE   2  (bases 1 to 5103)
  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: "Methods Mol
            Biol."; date: "2024"; volume: "2842:179-192. doi"; pages: " 
            10.1007/978-1-0716-4051-7_9"
COMMENT     Sequence Label: sgRNA_expression_vector
FEATURES             Location/Qualifiers
     source          1..5103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(27..46)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     81..100
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        156..404
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     156..176
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     327..346
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        432..507
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(597..1052)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(734..755)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     946..965
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1079..1183
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1185..1542
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1185..1376
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1208..1228)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1393..1528
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1577..2371
                     /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             1577..2371
                     /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(1631..1650)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2241..2260
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2559..2578)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2603..2650
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2979..3567
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3468..3487
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        3617..3920
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3921..4124
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4075..4095
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4289..4298
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4295..4972
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Strongin et al., 2007)"
                     /label=DsRed-Monomer
                     /note="mammalian codon-optimized"
                     /translation="MDNTEDVIKEFMQFKVRMEGSVNGHYFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYMKLSFPEGFTWERSMNFEDGGV
                     VEVQQDSSLQDGTFIYKVKFKGVNFPADGPVMQKKTAGWEPSTEKLYPQDGVLKGEISH
                     ALKLKDGGHYTCDFKTVYKAKKPVQLPGNHYVDSKLDITNHNEDYTVVEQYEHAEARHS
                     GSQ"
     polyA_signal    join(5093..5103,1..111)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
       61 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtctg
      121 ccttctggcc ttttgctggc cttttgctca catgtgaggg cctatttccc atgattcctt
      181 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
      241 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      301 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      361 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggggtc ttcggatcca
      421 tgcgaagacc tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
      481 cttgaaaaag tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa
      541 aataaggcta gtccgttttt agcgcgtgcg ccaattctgc agaccagaaa gctcgtaaat
      601 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
      661 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
      721 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
      781 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
      841 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
      901 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
      961 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     1021 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     1081 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1141 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     1201 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     1261 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     1321 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     1381 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     1441 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     1501 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     1561 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     1621 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     1681 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     1741 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     1801 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     1861 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     1921 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     1981 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     2041 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     2101 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     2161 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     2221 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     2281 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     2341 cttctatcga cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     2401 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     2461 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     2521 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     2581 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     2641 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     2701 agatcccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     2761 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     2821 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     2881 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2941 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3001 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3061 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3121 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3181 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3241 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3301 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3361 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3421 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3481 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3541 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttg gttcatagcc
     3601 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     3661 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     3721 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     3781 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     3841 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     3901 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     3961 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     4021 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     4081 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
     4141 agatccgcta gcgctaccgg actcagatct cgaccgagtt ccccgcgcca gcggggttaa
     4201 accgcgcccg ccagcttcga attctgcagt cgacggtacc gcgggccgag ttccccgcgc
     4261 cagcggggtt aaaccggatc caccggtcgc caccatggac aacaccgagg acgtcatcaa
     4321 ggagttcatg cagttcaagg tgcgcatgga gggctccgtg aacggccact acttcgagat
     4381 cgagggcgag ggcgagggca agccctacga gggcacccag accgccaagc tgcaggtgac
     4441 caagggcggc cccctgccct tcgcctggga catcctgtcc ccccagttcc agtacggctc
     4501 caaggcctac gtgaagcacc ccgccgacat ccccgactac atgaagctgt ccttccccga
     4561 gggcttcacc tgggagcgct ccatgaactt cgaggacggc ggcgtggtgg aggtgcagca
     4621 ggactcctcc ctgcaggacg gcaccttcat ctacaaggtg aagttcaagg gcgtgaactt
     4681 ccccgccgac ggccccgtaa tgcagaagaa aactgccggc tgggagccct ccaccgagaa
     4741 gctgtacccc caggacggcg tgctgaaggg cgagatctcc cacgccctga agctgaagga
     4801 cggcggccac tacacctgcg acttcaagac cgtgtacaag gccaagaagc ccgtgcagct
     4861 gcccggcaac cactacgtgg actccaagct ggacatcacc aaccacaacg aggactacac
     4921 cgtggtggag cagtacgagc acgccgaggc ccgccactcc ggctcccagt agcggccgcg
     4981 actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     5041 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     5101 tat
//
