LOCUS       sequence-476179-        3666 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO vector for the cloning of the SpCas9 tracrRNA-CS1 - AsCas12a 
            Direct Repeat (DR) v10 fragment into the pLCHKO_v4 hgRNA vector for 
            scCHyMErA-Seq experiments..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3666)
  TITLE     A Scalable Exon Deletion Platform Coupled To Single-Cell 
            Transcriptomics Reveals Exons Shaping Gene Expression Dynamics
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3666)
  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: 
            "Unpublished"
COMMENT     Sequence Label: Topo_SpCas9.tracrRNA-CS1_AsCas12a DRv10
FEATURES             Location/Qualifiers
     source          1..3666
                     /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"
     primer_bind     complement(554..573)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(555..573)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(580..597)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(580..596)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(589..611)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             734..1036
                     /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     915..934
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1385..2179
                     /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             1385..2179
                     /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(1439..1458)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2049..2068
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2379..2388
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2385..2759
                     /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      2897..3485
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3386..3405
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3639..3656
                     /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 gcccttaacg tctctaccta cgagagtagg
      361 aattcgcacc gactcggtgc cacttggcct tgctaggacc ggccttaaag cggccaagtt
      421 gataacggac tagccttatt tcaacttgct atgctgtttc cagcatagct ctgaaacaga
      481 gacgaagggc gaattctgca gatatccatc acactggcgg ccgctcgagc atgcatctag
      541 agggcccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg
      601 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
      661 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
      721 cctatacgta cggcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt
      781 tgtggatgta cagagtgata ttattgacac gccggggcga cggatggtga tccccctggc
      841 cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg
      901 ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg
      961 ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat
     1021 gttctgggga atataaatgt caggcatgag attatcaaaa aggatcttca cctagatcct
     1081 tttcacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg
     1141 ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct
     1201 tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc
     1261 tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc
     1321 gccaaggatc tgatggcgca ggggatcaag ctctgatcaa gagacaggat gaggatcgtt
     1381 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     1441 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     1501 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     1561 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     1621 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     1681 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     1741 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     1801 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     1861 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     1921 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     1981 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     2041 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     2101 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     2161 tcttgacgag ttcttctgaa ttattaacgc ttacaatttc ctgatgcggt attttctcct
     2221 tacgcatctg tgcggtattt cacaccgcat caggtggcac ttttcgggga aatgtgcgcg
     2281 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2341 aaccctgata aatgcttcaa taatagcacg tgaggagggc caccatggcc aagttgacca
     2401 gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc
     2461 ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg
     2521 tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg
     2581 tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact
     2641 tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt
     2701 tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac
     2761 acgtgctaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     2821 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2881 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2941 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3001 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     3061 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3121 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3181 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3241 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3301 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3361 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3421 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3481 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3541 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3601 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3661 cggaag
//
