LOCUS       sequence-181261-        3974 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 3974)
  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 3974)
  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..3974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(908..1282)
                     /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      1279..1288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1489..2283)
                     /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(1489..2283)
                     /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(1600..1619)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2210..2229
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(2632..2934)
                     /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(2734..2753)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3057..3079
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3071..3088
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3072..3088
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3095..3114
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3095..3113
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3253..3501
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3253..3273
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3424..3443
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3522..3597
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3609..3633)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(3718..3736)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3719..3736)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3754..3770)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3754..3770)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3767..3789)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3778..3794
                     /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(3802..3808,3809..3826,3827..3832))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3847..3868
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaagccca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 gcacgtgtca gtcctgctcc tcggccacga agtgcacgca gttgccggcc gggtcgcgca
      961 gggcgaactc ccgcccccac ggctgctcgc cgatctcggt catggccggc ccggaggcgt
     1021 cccggaagtt cgtggacacg acctccgacc actcggcgta cagctcgtcc aggccgcgca
     1081 cccacaccca ggccagggtg ttgtccggca ccacctggtc ctggaccgcg ctgatgaaca
     1141 gggtcacgtc gtcccggacc acaccggcga agtcgtcctc cacgaagtcc cgggagaacc
     1201 cgagccggtc ggtccagaac tcgaccgctc cggcgacgtc gcgcgcggtg agcaccggaa
     1261 cggcactggt caacttggcc atggtggccc tcctcacgtg ctattattga agcatttatc
     1321 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     1381 gggttccgcg cacatttccc cgaaaagtgc cacctgtatg cggtgtgaaa taccgcacag
     1441 atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg ttaataattc agaagaactc
     1501 gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac
     1561 gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc
     1621 tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg
     1681 gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc
     1741 gccgtcgggc atgctcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg
     1801 ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc
     1861 gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg
     1921 ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag
     1981 atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc
     2041 gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc
     2101 ttgcagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg
     2161 cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata
     2221 gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat
     2281 catgcgaaac gatcctcatc ctgtctcttg atcagagctt gatcccctgc gccatcagat
     2341 ccttggcggc gagaaagcca tccagtttac tttgcagggc ttcccaacct taccagaggg
     2401 cgccccagct ggcaattccg gttcgcttgc tgtccataaa accgcccagt ctagctatcg
     2461 ccatgtaagc ccactgcaag ctacctgctt tctctttgcg cttgcgtttt cccttgtcca
     2521 gatagcccag tagctgacat tcatccgggg tcagcaccgt ttctgcggac tggctttcta
     2581 cgtgaaaagg atctaggtga agatcctttt tgataatctc atgcctgaca tttatattcc
     2641 ccagaacatc aggttaatgg cgtttttgat gtcattttcg cggtggctga gatcagccac
     2701 ttcttccccg ataacggaga ccggcacact ggccatatcg gtggtcatca tgcgccagct
     2761 ttcatccccg atatgcacca ccgggtaaag ttcacgggag actttatctg acagcagacg
     2821 tgcactggcc agggggatca ccatccgtcg ccccggcgtg tcaataatat cactctgtac
     2881 atccacaaac agacgataac ggctctctct tttataggtg taaaccttaa actgccgtac
     2941 gtataggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     3001 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     3061 gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat agggcgaatt
     3121 gggccctcta gatgcatgct cgagcggccg ccagtgtgat ggatatctgc agaattcgcc
     3181 ctttgtacaa aaaagcaggc tttaaaggaa ccaattcagt cgactggatc cggtaccaag
     3241 gtcgggcagg aagagggcct atttcccatg attccttcat atttgcatat acgatacaag
     3301 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
     3361 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     3421 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     3481 ttgtggaaag gacgaaacac cgacttcaga taactacacc ggttttagag ctagaaatag
     3541 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     3601 ttttctagac ccagctttct tgtacaaagt tggcattaaa gggcgaattc cagcacactg
     3661 gcggccgtta ctagtggatc cgagctcggt accaagcttg atgcatagct tgagtattct
     3721 atagtgtcac ctaaatagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     3781 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     3841 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3901 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3961 gcgtattggg cgct
//
