LOCUS       sequence-319734-        4264 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate assembly and mammalian expression of dCas9 
            fusion-proteins plus gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4264)
  AUTHORS   Haynes KA, Priode JH
  TITLE     Rapid Single-Pot Assembly of Modular Chromatin Proteins for 
            Epigenetic Engineering.
  JOURNAL   Methods Mol Biol. 2023;2599:191-214. doi: 
            10.1007/978-1-0716-2847-8_14.
  PUBMED    36427151
REFERENCE   2  (bases 1 to 4264)
  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: "2023"; volume: "2599:191-214. doi"; pages: " 
            10.1007/978-1-0716-2847-8_14"
COMMENT     Sequence Label: GGDestX2-Amp
FEATURES             Location/Qualifiers
     source          1..4264
                     /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        266..341
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        438..723
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        725..1002
                     /label=chicken beta-actin promoter
     intron          1003..1230
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1242..1251
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1305..1322)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1311..1518
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   1527..1667
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1527..1656
                     /label=AAV2 ITR
     rep_origin      1742..2197
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1829..1848)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2039..2060
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2214..2233)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2333..2355
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2393..2411)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2479..2583
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2584..2652,2653..3444)
                     /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(2802..2821)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3615..4203
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4104..4123
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccc gagaccgagg tctcagtttt agagctagaa atagcaagtt aaaataaggc
      301 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt ttagagctag
      361 aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa
      421 atggctctag aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      481 aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa
      541 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      601 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac
      661 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      721 atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc
      781 ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg
      841 ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga
      901 ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg
      961 cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc
     1021 cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc
     1081 gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg
     1141 agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg
     1201 gagcacctgc ctgaaatcac tttttttcag gttggaccgg tgccaccatg gaggggtctt
     1261 cgagaagacc tactagctaa gaattcctag agctcgctga tcagcctcga ctgtgccttc
     1321 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     1381 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     1441 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa
     1501 tagcaggcat gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc
     1561 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     1621 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     1681 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     1741 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1801 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1861 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1921 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     1981 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2041 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     2101 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     2161 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     2221 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     2281 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     2341 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     2401 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     2461 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2521 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2581 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2641 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2701 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2761 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     2821 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     2881 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     2941 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3001 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3061 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3121 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3181 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3241 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga
     3301 agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3361 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3421 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3481 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3541 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3601 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3661 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3721 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     3781 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3841 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3901 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3961 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4021 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4081 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4141 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4201 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4261 atgt
//
