LOCUS       sequence-142759-        8727 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MCP-3XBFPnls.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8727)
  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 8727)
  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-MCP-3XBFPnls
FEATURES             Location/Qualifiers
     source          1..8727
                     /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      2521..2530
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2911..3621
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             3640..4350
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             4369..5079
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             5086..5106
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    5117..5705
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5170..5190)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5588..5599)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5780..6013
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6132..6150)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6218..6322
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6323..6391,6392..7183)
                     /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(6541..6560)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7354..7942
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7843..7862
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8096..8113
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8353..8374
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8389..8394,8395..8412,8413..8419)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8427..8443
                     /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     8432..8454
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8451..8467
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8451..8467
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8517..8536
                     /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 tccggttaag gtgaacgcgt gcaggctggc
     2521 gccaccatgg cttctaactt tactcagttc gttctcgtcg acaatggcgg aactggcgac
     2581 gtgactgtcg ccccaagcaa cttcgctaac gggatcgctg aatggatcag ctctaactcg
     2641 cgttcacagg cttacaaagt aacctgtagc gttcgtcaga gctctgcgca gaatcgcaaa
     2701 tacaccatca aagtcgaggt gcctaaaggc gcctggcgtt cgtacttaaa tatggaacta
     2761 accattccaa ttttcgccac gaattccgac tgcgagctta ttgttaaggc aatgcaaggt
     2821 ctcctaaaag atggaaaccc gattccctca gcaatcgcag caaactccgg catctacggg
     2881 ggatctggta gcggcgctgg atccggaagt atggtgtcta agggcgaaga gctgattaag
     2941 gagaacatgc acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc
     3001 acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc
     3061 gagggcggcc ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc
     3121 aagaccttca tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag
     3181 ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag
     3241 gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc
     3301 acatccaacg gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgagacg
     3361 ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc
     3421 gggagccatc tgatcgcaaa cgccaagacc acatatagat ccaagaaacc cgctaagaac
     3481 ctcaagatgc ctggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac
     3541 aacgaaacct acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc
     3601 aaactggggc acaagcttaa ttctggcggc cgctctggca tggtgtctaa gggcgaagag
     3661 ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga caaccatcac
     3721 ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc
     3781 aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac tagcttcctc
     3841 tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt caagcagtcc
     3901 ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc
     3961 gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg
     4021 gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccttc
     4081 accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat ggccctgaag
     4141 ctcgtgggcg ggagccatct gatcgcaaac gccaagacca catatagatc caagaaaccc
     4201 gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga aagaatcaag
     4261 gaggccaaca acgaaaccta cgtcgagcag cacgaggtgg cagtggccag atactgcgac
     4321 ctccctagca aactggggca caagcttaat ggtagcatcg atggtagcat ggtgtctaag
     4381 ggcgaagagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac
     4441 aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc
     4501 atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact
     4561 agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc
     4621 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
     4681 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag
     4741 atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg
     4801 gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg
     4861 gccctgaagc tcgtgggcgg gagccatctg atcgcaaacg ccaagaccac atatagatcc
     4921 aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa
     4981 agaatcaagg aggccaacaa cgaaacctac gtcgagcagc acgaggtggc agtggccaga
     5041 tactgcgacc tccctagcaa actggggcac aagcttaatc tcgagccaaa gaaaaagcgg
     5101 aaagtgtaat ctagataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     5161 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     5221 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     5281 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     5341 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     5401 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     5461 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     5521 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     5581 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     5641 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     5701 cccgcctgag atcctttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     5761 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
     5821 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     5881 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     5941 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     6001 aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     6061 aaatgaatat cagagagtga gaggcccggg ttaattaagg aaagggctag atcattcttg
     6121 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     6181 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     6241 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     6301 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     6361 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     6421 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     6481 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     6541 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     6601 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     6661 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     6721 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6781 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6841 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6901 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6961 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     7021 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     7081 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     7141 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     7201 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     7261 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     7321 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     7381 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     7441 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     7501 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     7561 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     7621 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     7681 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     7741 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     7801 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7861 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7921 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7981 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     8041 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     8101 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     8161 gccgattcat taatgcagca agctcatggc tgactaattt tttttattta tgcagaggcc
     8221 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     8281 ggcttttgca aaaagctccc cgtggcacga caggtttccc gactggaaag cgggcagtga
     8341 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     8401 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     8461 ctatgacatg attacgaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     8521 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc aactggataa ctcaagctaa
     8581 ccaaaatcat cccaaacttc ccaccccata ccctattacc actgccaatt acctgtggtt
     8641 tcatttactc taaacctgtg attcctctga attattttca ttttaaagaa attgtatttg
     8701 ttaaatatgt actacaaact tagtagt
//
