LOCUS       sequence-193145-        3973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for CRISPR gRNA targeting human CXCR4 
            exon 2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3973)
  AUTHORS   Heredia JD, Park J, Brubaker RJ, Szymanski SK, Gill KS, Procko E
  TITLE     Mapping Interaction Sites on Human Chemokine Receptors by Deep 
            Mutational Scanning.
  JOURNAL   J Immunol. 2018 Apr 20. pii: jimmunol.1800343. doi: 
            10.4049/jimmunol.1800343.
  PUBMED    29678950
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"; journalName: "J Immunol. 
            2018 Apr 20. pii: jimmunol.1800343. doi: 10.4049/jimmunol.1800343."
COMMENT     Sequence Label: gRNA-CXCR4(90)
FEATURES             Location/Qualifiers
     source          1..3973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        182..430
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     182..202
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     353..372
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        451..526
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(538..562)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(647..665)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(648..665)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(683..699)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(683..699)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(696..718)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    707..723
                     /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(731..737,738..755,756..761))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    776..797
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(914..931)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1085..1673)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1165..1184)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1811..2185)
                     /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      2182..2191
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2391..3185)
                     /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(2391..3185)
                     /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(2502..2521)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3112..3131
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3534..3836)
                     /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(3636..3655)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     join(3959..3973,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(3973,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggccctctag
       61 atgcatgctc gagcggccgc cagtgtgatg gatatctgca gaattcgccc tttgtacaaa
      121 aaagcaggct ttaaaggaac caattcagtc gactggatcc ggtaccaagg tcgggcagga
      181 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
      241 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
      301 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
      361 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
      421 acgaaacacc gacttcagat aactacaccg gttttagagc tagaaatagc aagttaaaat
      481 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttctagacc
      541 cagctttctt gtacaaagtt ggcattaaag ggcgaattcc agcacactgg cggccgttac
      601 tagtggatcc gagctcggta ccaagcttga tgcatagctt gagtattcta tagtgtcacc
      661 taaatagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
      721 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
      781 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
      841 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
      901 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
      961 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     1021 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     1081 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     1141 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     1201 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     1261 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     1321 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     1381 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     1441 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     1501 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     1561 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     1621 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     1681 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     1741 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     1801 ttagcacgtg tcagtcctgc tcctcggcca cgaagtgcac gcagttgccg gccgggtcgc
     1861 gcagggcgaa ctcccgcccc cacggctgct cgccgatctc ggtcatggcc ggcccggagg
     1921 cgtcccggaa gttcgtggac acgacctccg accactcggc gtacagctcg tccaggccgc
     1981 gcacccacac ccaggccagg gtgttgtccg gcaccacctg gtcctggacc gcgctgatga
     2041 acagggtcac gtcgtcccgg accacaccgg cgaagtcgtc ctccacgaag tcccgggaga
     2101 acccgagccg gtcggtccag aactcgaccg ctccggcgac gtcgcgcgcg gtgagcaccg
     2161 gaacggcact ggtcaacttg gccatggtgg ccctcctcac gtgctattat tgaagcattt
     2221 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     2281 taggggttcc gcgcacattt ccccgaaaag tgccacctga tgcggtgtga aataccgcac
     2341 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaataat tcagaagaac
     2401 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc
     2461 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac
     2521 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag
     2581 cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc
     2641 tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga
     2701 tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc
     2761 tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc
     2821 cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg
     2881 agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg
     2941 tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg
     3001 tcttgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc
     3061 tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca
     3121 tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca
     3181 atcatgcgaa acgatcctca tcctgtctct tgatcagagc ttgatcccct gcgccatcag
     3241 atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac cttaccagag
     3301 ggcgccccag ctggcaattc cggttcgctt gctgtccata aaaccgccca gtctagctat
     3361 cgccatgtaa gcccactgca agctacctgc tttctctttg cgcttgcgtt ttcccttgtc
     3421 cagatagccc agtagctgac attcatccgg ggtcagcacc gtttctgcgg actggctttc
     3481 tacgtgaaaa ggatctaggt gaagatcctt tttgataatc tcatgcctga catttatatt
     3541 ccccagaaca tcaggttaat ggcgtttttg atgtcatttt cgcggtggct gagatcagcc
     3601 acttcttccc cgataacgga gaccggcaca ctggccatat cggtggtcat catgcgccag
     3661 ctttcatccc cgatatgcac caccgggtaa agttcacggg agactttatc tgacagcaga
     3721 cgtgcactgg ccagggggat caccatccgt cgccccggcg tgtcaataat atcactctgt
     3781 acatccacaa acagacgata acggctctct cttttatagg tgtaaacctt aaactgccgt
     3841 acgtataggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     3901 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     3961 cagtcacgac gtt
//
