LOCUS       sequence-326713-        5297 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression vector containing the miRFP670 fluorescent marker to
            target the region downstream of the SRR134 SOX2 enhancer in human 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5297)
  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 5297)
  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_SRR134.3_miRFP670
FEATURES             Location/Qualifiers
     source          1..5297
                     /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"
     misc_RNA        complement(378..453)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(475..723)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(533..552)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(703..723)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(862..881)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(863..881)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(888..905)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(888..904)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(897..919)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1042..1344
                     /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     1223..1242
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1693..2487
                     /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             1693..2487
                     /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(1747..1766)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2357..2376
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     enhancer        2643..2946
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2947..3150
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3101..3121
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    3173..3253
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      3255..3264
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3261..4208
                     /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    4330..4451
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4367..4386)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4421..4440
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4528..5116
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5017..5036
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5270..5287
                     /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 cttgctattt ctagctctaa aacactattc gtcctcatcc ttccggtgtt
      481 tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag
      541 catatgatag tccattttaa aacataattt taaaactgca aactacccaa gaaattatta
      601 ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattctaat
      661 tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc
      721 ctcttcctgc ccgaccttgg taccggatcc agtcgactga attggttcct ttaaagcctg
      781 cttttttgta caaagggcga attctgcaga tatccatcac actggcggcc gctcgagcat
      841 gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
      901 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      961 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     1021 ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga gagccgttat
     1081 cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg gatggtgatc
     1141 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg
     1201 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc
     1261 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt
     1321 aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag gatcttcacc
     1381 tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc
     1441 agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc
     1501 agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa
     1561 ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct
     1621 ttcttgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga gacaggatga
     1681 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     1741 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     1801 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     1861 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     1921 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     1981 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     2041 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     2101 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     2161 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     2221 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     2281 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     2341 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     2401 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     2461 tatcgccttc ttgacgagtt cttctgaatt attaacgctt acaatttcct gatgcggtat
     2521 tttctcctta cgcatctgtg cggtatttca caccgcatca ggtggcactt ttcggggaaa
     2581 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2641 gacgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     2701 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     2761 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     2821 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     2881 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     2941 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     3001 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     3061 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     3121 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
     3181 taccggactc agatctcgag ctcaagcttc gaattctgca gtcgacggta ccgcgggccc
     3241 gggatccacc ggtcgccacc atggtagcag gtcatgcctc tggcagcccc gcattcggga
     3301 ccgcctctca ttcgaattgc gaacatgaag agatccacct cgccggctcg atccagccgc
     3361 atggcgcgct tctggtcgtc agcgaacatg atcatcgcgt catccaggcc agcgccaacg
     3421 ccgcggaatt tctgaatctc ggaagcgtac tcggcgttcc gctcgccgag atcgacggcg
     3481 atctgttgat caagatcctg ccgcatctcg atcccaccgc cgaaggcatg ccggtcgcgg
     3541 tgcgctgccg gatcggcaat ccctctacgg agtactgcgg tctgatgcat cggcctccgg
     3601 aaggcgggct gatcatcgaa ctcgaacgtg ccggcccgtc gatcgatctg tcaggcacgc
     3661 tggcgccggc gctggagcgg atccgcacgg cgggttcact gcgcgcgctg tgcgatgaca
     3721 ccgtgctgct gtttcagcag tgcaccggct acgaccgggt gatggtgtat cgtttcgatg
     3781 agcaaggcca cggcctggta ttctccgagt gccatgtgcc tgggctcgaa tcctatttcg
     3841 gcaaccgcta tccgtcgtcg actgtcccgc agatggcgcg gcagctgtac gtgcggcagc
     3901 gcgtccgcgt gctggtcgac gtcacctatc agccggtgcc gctggagccg cggctgtcgc
     3961 cgctgaccgg gcgcgatctc gacatgtcgg gctgcttcct gcgctcgatg tcgccgtgcc
     4021 atctgcagtt cctgaaggac atgggcgtgc gcgccaccct ggcggtgtcg ctggtggtcg
     4081 gcggcaagct gtggggcctg gttgtctgtc accattatct gccgcgcttc atccgtttcg
     4141 agctgcgggc gatctgcaaa cggctcgccg aaaggatcgc gacgcggatc accgcgcttg
     4201 agagctaagc ggccgcgact ctagatcata atcagccata ccacatttgt agaggtttta
     4261 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     4321 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4381 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4441 aatgtatctt atcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4501 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4561 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4621 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4681 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4741 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4801 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4861 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4921 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4981 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5041 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5101 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5161 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5221 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     5281 gagcgaggaa gcggaag
//
