LOCUS       sequence-142762-        8440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N22p-3XRFPnls.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8440)
  AUTHORS   Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T
  TITLE     Multiplexed labeling of genomic loci with dCas9 and engineered 
            sgRNAs using CRISPRainbow.
  JOURNAL   Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.
  PUBMED    27088723
REFERENCE   2  (bases 1 to 8440)
  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: "Nat 
            Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526."
COMMENT     Sequence Label: pHAGE-EFS-N22p-3XRFPnls
FEATURES             Location/Qualifiers
     source          1..8440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2265..2476
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2522..2531
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2633..3340
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     CDS             3359..4066
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     CDS             4085..4792
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     CDS             4799..4819
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    4830..5418
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4883..4903)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5301..5312)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5493..5726
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5845..5863)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5931..6035
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6036..6104,6105..6896)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6254..6273)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7067..7655
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7556..7575
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7809..7826
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8066..8087
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8102..8107,8108..8125,8126..8132)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8140..8156
                     /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     8145..8167
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8164..8180
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8164..8180
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8230..8249
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttgacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagcgat
     2221 gctagctgat gcgggccctt cgagtggctc cggtgcccgt cagtgggcag agcgcacatc
     2281 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     2341 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     2401 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     2461 tgccgccaga acacaggtgt cgtgacgcgg ttccggttaa ggtgaacgcg tgcaggctgg
     2521 cgccaccatg ggtaatgctc ggacccggcg aagagagagg cgggctgaga agcaggcaca
     2581 gtggaaggct gcaaacggtg gcggaggtac tagtggctct ggatccggaa gtatggtgag
     2641 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     2701 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     2761 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     2821 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     2881 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     2941 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tcactgcagg acggcgagtt
     3001 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     3061 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     3121 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     3181 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     3241 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgctga
     3301 gggccgccac tccaccggcg gcatggacga gctgtacaag tctggcggcc gctctggcat
     3361 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     3421 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     3481 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     3541 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     3601 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     3661 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctcac tgcaggacgg
     3721 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     3781 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     3841 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     3901 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     3961 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     4021 cgctgagggc cgccactcca ccggcggcat ggacgagctg tacaagggta gcatcgatgg
     4081 tagcatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     4141 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     4201 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     4261 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     4321 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     4381 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctcactgca
     4441 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     4501 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     4561 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     4621 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     4681 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     4741 cgaacgcgct gagggccgcc actccaccgg cggcatggac gagctgtaca agctcgagcc
     4801 aaagaaaaag cggaaagtgt aatctagata atcaacctct ggattacaaa atttgtgaaa
     4861 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     4921 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     4981 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     5041 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     5101 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     5161 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     5221 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     5281 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     5341 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     5401 tttgggccgc ctccccgcct gagatccttt aagaccaatg acttacaagg cagctgtaga
     5461 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     5521 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
     5581 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     5641 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     5701 ttagtcagtg tggaaaatct ctagcagtag tagttcatgt catcttatta ttcagtattt
     5761 ataacttgca aagaaatgaa tatcagagag tgagaggccc gggttaatta aggaaagggc
     5821 tagatcattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     5881 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5941 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     6001 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     6061 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     6121 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     6181 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     6241 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     6301 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     6361 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     6421 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6481 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6541 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6601 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6661 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6721 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6781 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6841 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6901 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6961 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     7021 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     7081 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     7141 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     7201 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     7261 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     7321 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     7381 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     7441 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     7501 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7561 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7621 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7681 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     7741 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     7801 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     7861 tccccgcgcg ttggccgatt cattaatgca gcaagctcat ggctgactaa ttttttttat
     7921 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     7981 ttttggaggc ctaggctttt gcaaaaagct ccccgtggca cgacaggttt cccgactgga
     8041 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     8101 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     8161 acacaggaaa cagctatgac atgattacga atttcacaaa taaagcattt ttttcactgc
     8221 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaactgga
     8281 taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt accactgcca
     8341 attacctgtg gtttcattta ctctaaacct gtgattcctc tgaattattt tcattttaaa
     8401 gaaattgtat ttgttaaata tgtactacaa acttagtagt
//
