LOCUS       sequence-327833-        5237 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Empty gRNA cloning vector, contains a miRFP670 fluorescent marker 
            for FACS sorting after transfection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5237)
  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 5237)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_miRFP670
FEATURES             Location/Qualifiers
     source          1..5237
                     /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"
     enhancer        2583..2886
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2887..3090
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3041..3061
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    3113..3193
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      3195..3204
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3201..4148
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     polyA_signal    4270..4391
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4307..4326)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4361..4380
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4468..5056
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4957..4976
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5210..5227
                     /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 gacgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     2641 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     2701 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     2761 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     2821 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     2881 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     2941 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     3001 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     3061 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
     3121 taccggactc agatctcgag ctcaagcttc gaattctgca gtcgacggta ccgcgggccc
     3181 gggatccacc ggtcgccacc atggtagcag gtcatgcctc tggcagcccc gcattcggga
     3241 ccgcctctca ttcgaattgc gaacatgaag agatccacct cgccggctcg atccagccgc
     3301 atggcgcgct tctggtcgtc agcgaacatg atcatcgcgt catccaggcc agcgccaacg
     3361 ccgcggaatt tctgaatctc ggaagcgtac tcggcgttcc gctcgccgag atcgacggcg
     3421 atctgttgat caagatcctg ccgcatctcg atcccaccgc cgaaggcatg ccggtcgcgg
     3481 tgcgctgccg gatcggcaat ccctctacgg agtactgcgg tctgatgcat cggcctccgg
     3541 aaggcgggct gatcatcgaa ctcgaacgtg ccggcccgtc gatcgatctg tcaggcacgc
     3601 tggcgccggc gctggagcgg atccgcacgg cgggttcact gcgcgcgctg tgcgatgaca
     3661 ccgtgctgct gtttcagcag tgcaccggct acgaccgggt gatggtgtat cgtttcgatg
     3721 agcaaggcca cggcctggta ttctccgagt gccatgtgcc tgggctcgaa tcctatttcg
     3781 gcaaccgcta tccgtcgtcg actgtcccgc agatggcgcg gcagctgtac gtgcggcagc
     3841 gcgtccgcgt gctggtcgac gtcacctatc agccggtgcc gctggagccg cggctgtcgc
     3901 cgctgaccgg gcgcgatctc gacatgtcgg gctgcttcct gcgctcgatg tcgccgtgcc
     3961 atctgcagtt cctgaaggac atgggcgtgc gcgccaccct ggcggtgtcg ctggtggtcg
     4021 gcggcaagct gtggggcctg gttgtctgtc accattatct gccgcgcttc atccgtttcg
     4081 agctgcgggc gatctgcaaa cggctcgccg aaaggatcgc gacgcggatc accgcgcttg
     4141 agagctaagc ggccgcgact ctagatcata atcagccata ccacatttgt agaggtttta
     4201 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     4261 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4321 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4381 aatgtatctt atcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4441 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4501 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4561 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4621 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4681 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4741 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4801 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4861 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4921 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4981 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5041 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5101 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5161 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     5221 gagcgaggaa gcggaag
//
