LOCUS       sequence-194499-        7975 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes dCas9(N) fused to CXCR4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7975)
  AUTHORS   Baeumler TA, Ahmed AA, Fulga TA
  TITLE     Engineering Synthetic Signaling Pathways with Programmable 
            dCas9-Based Chimeric Receptors.
  JOURNAL   Cell Rep. 2017 Sep 12;20(11):2639-2653. doi: 
            10.1016/j.celrep.2017.08.044.
  PUBMED    28903044
REFERENCE   2  (bases 1 to 7975)
  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"; doi: 
            "10.1016/j.celrep.2017.08"; journalName: "Cell Rep"; date: 
            "2017-09-12- 12"; volume: "20"; issue: "11"; pages: "2639-2653"
COMMENT     Sequence Label: pCXCR4_TCS(Q'L)_HA-dCas9(N)_P2A-Puro-WPRE
FEATURES             Location/Qualifiers
     source          1..7975
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        12..297
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        299..576
                     /label=chicken beta-actin promoter
     intron          577..805
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             2088..2114
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2130..2153,2154..2156,2157..3731)
                     /codon_start=1
                     /product="N-terminal portion of catalytically inactive 
                     Streptococcus pyogenes dCas9 carrying the D10A mutation 
                     (Zetsche et al., 2015)"
                     /label=dCas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMR"
     CDS             3762..3818
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(3825..3844)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4321..4341
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4458..4478)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4876..4887)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5018..5035)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5024..5231
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5240..5380
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5240..5369
                     /label=AAV2 ITR
     rep_origin      5455..5910
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5542..5561)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5752..5773
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5927..5946)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6046..6068
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6106..6124)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6192..6296
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6297..6365,6366..7157)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6515..6534)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7328..7916
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7817..7836
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
       61 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg
      121 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      181 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc
      241 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      301 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      361 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      421 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      481 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      541 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg
      601 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt
      661 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa
      721 gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca
      781 cctgcctgaa atcacttttt ttcaggttgg accggtgcgc caccatgaag acgatcatcg
      841 ccctgagcta catcttctgc ctggtattcg ccgactacaa ggacgatgat gacgccagca
      901 tcgatatgga ggggatatca atctacacat cagataatta cacggaagag atgggttccg
      961 gcgattacga ctctatgaaa gagccgtgtt ttagagagga aaacgcgaac tttaacaaga
     1021 tctttctccc caccatctac agcatcatct ttctcacagg catcgtaggg aacggcctgg
     1081 tcatcctggt gatgggatac caaaagaagc tgaggtcaat gaccgacaag tataggctcc
     1141 atctgtccgt ggccgacctc ctgtttgtga ttaccctgcc tttttgggca gttgacgctg
     1201 tcgctaattg gtacttcggc aacttcctct gtaaggcagt gcacgttatc tacactgtga
     1261 atctttatag ttccgtcttg atcttggcct ttatcagtct cgacaggtat ttggcgattg
     1321 tgcacgctac caactcacaa cgacctagaa aactcctggc tgagaaggtg gtttacgttg
     1381 gtgtgtggat tccagctctc ctgttgacaa taccagactt cattttcgct aatgtgagcg
     1441 aggccgatga cagatacatt tgtgaccgat tttacccaaa cgatctgtgg gtagtggtat
     1501 ttcagttcca acacattatg gtcgggctga tcttgcccgg cattgtcata ctgtcttgct
     1561 actgcatcat tatttctaag ctgtcacact caaaaggcca ccaaaagagg aaggctctga
     1621 aaacaacggt gatcctgata ctggccttct tcgcatgttg gctgccctac tatatcggca
     1681 tcagcattga ctcatttata ctcctggaaa ttatcaagca gggctgcgag ttcgagaaca
     1741 ccgttcataa gtggatttct ataaccgagg ccctcgcctt ctttcactgt tgtttgaatc
     1801 cgattctcta cgcgtttctt ggcgccaaat ttaaaacaag cgcccaacat gcactgacat
     1861 cagtgtctag ggggagctct ctgaaaatcc tctccaaggg aaaacgaggc ggacatagca
     1921 gtgtcagcac tgagtccgaa tccagctcat ttcatagctc tatcgatacc ggtggacgca
     1981 ccccacccag cctgggtccc caagatgagt cctgcaccac cgccagctcc tccctggcca
     2041 aggacacttc atcgaccggt gagaacctgt acttccagct aactagttac ccatacgacg
     2101 taccagatta cgctggaggt tctggatccg acaagaagta cagcatcggc ctggccatcg
     2161 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     2221 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     2281 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     2341 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     2401 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     2461 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     2521 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     2581 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     2641 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     2701 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     2761 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     2821 agaagaagaa tggcctgttc ggcaacctga ttgccctgag cctgggcctg acccccaact
     2881 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     2941 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     3001 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     3061 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     3121 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     3181 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     3241 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     3301 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     3361 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     3421 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     3481 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     3541 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     3601 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     3661 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     3721 agggaatgag aggtggcggt ggctcgggta ccggatctgg agccaccaat ttttctctgc
     3781 tgaagcaggc tggtgatgtt gaagaaaacc ctggacctga attcatgacc gagtacaagc
     3841 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     3901 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     3961 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     4021 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     4081 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     4141 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     4201 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     4261 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     4321 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     4381 aaggaccgcg cacctggtgc atgacccgca aggggtaact aagtaaggat cgatccaaga
     4441 tatcgtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     4501 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     4561 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     4621 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     4681 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     4741 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag
     4801 ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     4861 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     4921 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     4981 gccgcctccc cgccaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     5041 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     5101 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     5161 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg
     5221 catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     5281 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     5341 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     5401 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     5461 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     5521 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     5581 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     5641 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     5701 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     5761 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
     5821 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5881 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     5941 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     6001 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     6061 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     6121 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     6181 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     6241 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     6301 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     6361 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     6421 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     6481 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     6541 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     6601 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     6661 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     6721 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6781 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6841 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6901 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6961 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg
     7021 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     7081 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     7141 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     7201 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     7261 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     7321 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     7381 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     7441 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     7501 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     7561 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     7621 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     7681 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     7741 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     7801 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     7861 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     7921 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacat
//
