LOCUS       sequence-444969-        3973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9 close to the ATG of giantin/GOLGB1 gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3973)
  AUTHORS   Fourriere L, Divoux S, Roceri M, Perez F, Boncompain G
  TITLE     Microtubule-independent secretion requires functional maturation of 
            Golgi elements.
  JOURNAL   J Cell Sci. 2016 Sep 1;129(17):3238-50. doi: 10.1242/jcs.188870. 
            Epub 2016 Jul 13.
  PUBMED    27411366
REFERENCE   2  (bases 1 to 3973)
  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"; doi: 
            "10.1242/jcs.188870"; journalName: "J Cell Sci"; date: "2016-09-1- 
            1"; volume: "129"; issue: "17"; pages: "3238-50"
COMMENT     Sequence Label: gRNA giantin/GOLGB1
FEATURES             Location/Qualifiers
     source          1..3973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(138..155)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(309..897)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(389..408)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1035..1409)
                     /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      1406..1415
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1615..2409)
                     /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(1615..2409)
                     /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(1726..1745)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2336..2355
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(2758..3060)
                     /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(2860..2879)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3183..3205
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3197..3214
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3198..3214
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3221..3240
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3221..3239
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3379..3627
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3379..3399
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3550..3569
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3648..3723
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3735..3759)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(3844..3862)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3845..3862)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3880..3896)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3880..3896)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3893..3915)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3904..3920
                     /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(3928..3934,3935..3952,3953..3958))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(3973,1..21)
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
       61 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
      121 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
      181 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
      241 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
      301 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
      361 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
      421 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
      481 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
      541 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
      601 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
      661 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
      721 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
      781 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
      841 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
      901 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
      961 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     1021 agttttagca cgtgtcagtc ctgctcctcg gccacgaagt gcacgcagtt gccggccggg
     1081 tcgcgcaggg cgaactcccg cccccacggc tgctcgccga tctcggtcat ggccggcccg
     1141 gaggcgtccc ggaagttcgt ggacacgacc tccgaccact cggcgtacag ctcgtccagg
     1201 ccgcgcaccc acacccaggc cagggtgttg tccggcacca cctggtcctg gaccgcgctg
     1261 atgaacaggg tcacgtcgtc ccggaccaca ccggcgaagt cgtcctccac gaagtcccgg
     1321 gagaacccga gccggtcggt ccagaactcg accgctccgg cgacgtcgcg cgcggtgagc
     1381 accggaacgg cactggtcaa cttggccatg gtggccctcc tcacgtgcta ttattgaagc
     1441 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     1501 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgatgcggt gtgaaatacc
     1561 gcacagatgc gtaaggagaa aataccgcat caggaaattg taagcgttaa taattcagaa
     1621 gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta
     1681 aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc
     1741 caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga
     1801 aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag
     1861 atcctcgccg tcgggcatgc tcgccttgag cctggcgaac agttcggctg gcgcgagccc
     1921 ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc
     1981 tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg
     2041 cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga
     2101 caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac
     2161 aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc
     2221 ctcgtcttgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg
     2281 cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca
     2341 gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg
     2401 ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gagcttgatc ccctgcgcca
     2461 tcagatcctt ggcggcaaga aagccatcca gtttactttg cagggcttcc caaccttacc
     2521 agagggcgcc ccagctggca attccggttc gcttgctgtc cataaaaccg cccagtctag
     2581 ctatcgccat gtaagcccac tgcaagctac ctgctttctc tttgcgcttg cgttttccct
     2641 tgtccagata gcccagtagc tgacattcat ccggggtcag caccgtttct gcggactggc
     2701 tttctacgtg aaaaggatct aggtgaagat cctttttgat aatctcatgc ctgacattta
     2761 tattccccag aacatcaggt taatggcgtt tttgatgtca ttttcgcggt ggctgagatc
     2821 agccacttct tccccgataa cggagaccgg cacactggcc atatcggtgg tcatcatgcg
     2881 ccagctttca tccccgatat gcaccaccgg gtaaagttca cgggagactt tatctgacag
     2941 cagacgtgca ctggccaggg ggatcaccat ccgtcgcccc ggcgtgtcaa taatatcact
     3001 ctgtacatcc acaaacagac gataacggct ctctctttta taggtgtaaa ccttaaactg
     3061 ccgtacgtat aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     3121 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     3181 ttcccagtca cgacgttgta aaacgacggc cagtgaattg taatacgact cactataggg
     3241 cgaattgggc cctctagatg catgctcgag cggccgccag tgtgatggat atctgcagaa
     3301 ttcgcccttt gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac tggatccggt
     3361 accaaggtcg ggcaggaaga gggcctattt cccatgattc cttcatattt gcatatacga
     3421 tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta
     3481 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     3541 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     3601 tatatcttgt ggaaaggacg aaacaccgaa atgctgagcc gattatcgtt ttagagctag
     3661 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     3721 tgcttttttt ctagacccag ctttcttgta caaagttggc attaaagggc gaattccagc
     3781 acactggcgg ccgttactag tggatccgag ctcggtacca agcttgatgc atagcttgag
     3841 tattctatag tgtcacctaa atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     3901 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     3961 ctggggtgcc taa
//
