LOCUS       sequence-222143-        8728 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 8728)
  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 8728)
  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..8728
                     /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             2912..3622
                     /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             3641..4351
                     /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             4370..5080
                     /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             5087..5107
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    5118..5706
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5171..5191)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5589..5600)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5781..6014
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6133..6151)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6219..6323
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6324..6392,6393..7184)
                     /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(6542..6561)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7355..7943
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7844..7863
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8097..8114
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8354..8375
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8390..8395,8396..8413,8414..8420)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8428..8444
                     /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     8433..8455
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8452..8468
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8452..8468
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8518..8537
                     /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 gcttctaact ttactcagtt cgttctcgtc gacaatggcg gaactggcga
     2581 cgtgactgtc gccccaagca acttcgctaa cgggatcgct gaatggatca gctctaactc
     2641 gcgttcacag gcttacaaag taacctgtag cgttcgtcag agctctgcgc agaatcgcaa
     2701 atacaccatc aaagtcgagg tgcctaaagg cgcctggcgt tcgtacttaa atatggaact
     2761 aaccattcca attttcgcca cgaattccga ctgcgagctt attgttaagg caatgcaagg
     2821 tctcctaaaa gatggaaacc cgattccctc agcaatcgca gcaaactccg gcatctacgg
     2881 gggatctggt agcggcgctg gatccggaag tatggtgtct aagggcgaag agctgattaa
     2941 ggagaacatg cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg
     3001 cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt
     3061 cgagggcggc cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag
     3121 caagaccttc atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga
     3181 gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca
     3241 ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt
     3301 cacatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgagac
     3361 gctgtacccc gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg
     3421 cgggagccat ctgatcgcaa acgccaagac cacatataga tccaagaaac ccgctaagaa
     3481 cctcaagatg cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa
     3541 caacgaaacc tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag
     3601 caaactgggg cacaagctta attctggcgg ccgctctggc atggtgtcta agggcgaaga
     3661 gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtgg acaaccatca
     3721 cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat
     3781 caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta ctagcttcct
     3841 ctacggcagc aagaccttca tcaaccacac ccagggcatc cccgacttct tcaagcagtc
     3901 cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac
     3961 cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg
     4021 ggtgaacttc acatccaacg gccctgtgat gcagaagaaa acactcggct gggaggcctt
     4081 caccgagacg ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca tggccctgaa
     4141 gctcgtgggc gggagccatc tgatcgcaaa cgccaagacc acatatagat ccaagaaacc
     4201 cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac tacagactgg aaagaatcaa
     4261 ggaggccaac aacgaaacct acgtcgagca gcacgaggtg gcagtggcca gatactgcga
     4321 cctccctagc aaactggggc acaagcttaa tggtagcatc gatggtagca tggtgtctaa
     4381 gggcgaagag ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga
     4441 caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac
     4501 catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac
     4561 tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt
     4621 caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg
     4681 cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa
     4741 gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg
     4801 ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat
     4861 ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac gccaagacca catatagatc
     4921 caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga
     4981 aagaatcaag gaggccaaca acgaaaccta cgtcgagcag cacgaggtgg cagtggccag
     5041 atactgcgac ctccctagca aactggggca caagcttaat ctcgagccaa agaaaaagcg
     5101 gaaagtgtaa tctagataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     5161 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     5221 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     5281 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     5341 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     5401 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     5461 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     5521 cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     5581 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     5641 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     5701 ccccgcctga gatcctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     5761 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct
     5821 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     5881 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     5941 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     6001 gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa
     6061 gaaatgaata tcagagagtg agaggcccgg gttaattaag gaaagggcta gatcattctt
     6121 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     6181 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     6241 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     6301 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     6361 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     6421 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     6481 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     6541 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     6601 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     6661 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     6721 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     6781 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6841 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6901 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6961 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     7021 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     7081 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     7141 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     7201 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     7261 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     7321 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     7381 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     7441 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     7501 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     7561 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     7621 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     7681 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     7741 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     7801 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7861 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7921 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7981 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     8041 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     8101 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     8161 ggccgattca ttaatgcagc aagctcatgg ctgactaatt ttttttattt atgcagaggc
     8221 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     8281 aggcttttgc aaaaagctcc ccgtggcacg acaggtttcc cgactggaaa gcgggcagtg
     8341 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     8401 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     8461 gctatgacat gattacgaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     8521 gtttgtccaa actcatcaat gtatcttatc atgtctggat caactggata actcaagcta
     8581 accaaaatca tcccaaactt cccaccccat accctattac cactgccaat tacctgtggt
     8641 ttcatttact ctaaacctgt gattcctctg aattattttc attttaaaga aattgtattt
     8701 gttaaatatg tactacaaac ttagtagt
//
