LOCUS       sequence-326714-        4914 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty gRNA cloning vector, contains a tagBFP fluorescent marker for 
            FACS sorting after transfection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4914)
  AUTHORS   Abatti LE, Lado-Fernandez P, Huynh L, Collado M, Hoffman MM, 
            Mitchell JA
  TITLE     Epigenetic reprogramming of a distal developmental enhancer cluster 
            drives SOX2 overexpression in breast and lung adenocarcinoma.
  JOURNAL   Nucleic Acids Res. 2023 Sep 22:gkad734. doi: 10.1093/nar/gkad734.
  PUBMED    37738673
REFERENCE   2  (bases 1 to 4914)
  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: "Nucleic 
            Acids Res. 2023 Sep 22:gkad734. doi: 10.1093/nar/gkad734."
COMMENT     Sequence Label: gRNA_CloningVector_tagBFP
FEATURES             Location/Qualifiers
     source          1..4914
                     /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"
     protein_bind    342..366
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(473..492)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(643..663)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(802..821)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(803..821)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(828..845)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(828..844)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(837..859)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             982..1284
                     /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     1163..1182
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1633..2427
                     /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             1633..2427
                     /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(1687..1706)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2297..2316
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        2886..3089
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3040..3060
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             3124..3825
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    3947..4068
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3984..4003)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4038..4057
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4145..4733
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4634..4653
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4887..4904
                     /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 gccctttaat gccaactttg tacaagaaag
      361 ctgggtctag aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc
      421 cttattttaa cttaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag
      481 catatgatag tccattttaa aacataattt taaaactgca aactacccaa gaaattatta
      541 ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattctaat
      601 tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc
      661 ctcttcctgc ccgaccttgg taccggatcc agtcgactga attggttcct ttaaagcctg
      721 cttttttgta caaagggcga attctgcaga tatccatcac actggcggcc gctcgagcat
      781 gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
      841 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      901 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      961 ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga gagccgttat
     1021 cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg gatggtgatc
     1081 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg
     1141 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc
     1201 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt
     1261 aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag gatcttcacc
     1321 tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc
     1381 agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc
     1441 agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa
     1501 ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct
     1561 ttcttgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga gacaggatga
     1621 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     1681 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     1741 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     1801 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     1861 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     1921 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     1981 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     2041 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     2101 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     2161 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     2221 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     2281 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     2341 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     2401 tatcgccttc ttgacgagtt cttctgaatt attaacgctt acaatttcct gatgcggtat
     2461 tttctcctta cgcatctgtg cggtatttca caccgcatca ggtggcactt ttcggggaaa
     2521 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2581 gacgttaata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     2641 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     2701 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     2761 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     2821 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     2881 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     2941 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     3001 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     3061 tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgcc
     3121 accatgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg
     3181 gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag
     3241 accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct
     3301 actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc
     3361 ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg
     3421 ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc
     3481 aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc
     3541 tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg cagaaacgac
     3601 atggccctga agctcgtggg cgggagccat ctgatcgcaa acatcaagac cacatataga
     3661 tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga ctacagactg
     3721 gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt ggcagtggcc
     3781 agatactgcg acctccctag caaactgggg cacaagctta attaggcggc cgcgactcta
     3841 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     3901 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     3961 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4021 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgaccaaaa
     4081 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4141 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4201 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4261 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     4321 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4381 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4441 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4501 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4561 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4621 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4681 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4741 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4801 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4861 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaag
//
