LOCUS       sequence-311036-        8168 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tagging clonal populations with NES EYFP fluorescent protein. Adding
            NES to EYFP WC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8168)
  AUTHORS   Kaufman T, Nitzan E, Firestein N, Ginzberg MB, Iyengar S, Patel N, 
            Ben-Hamo R, Porat Z, Hunter J, Hilfinger A, Rotter V, Kafri R, 
            Straussman R
  TITLE     Visual barcodes for clonal-multiplexing of live microscopy-based 
            assays.
  JOURNAL   Nat Commun. 2022 May 18;13(1):2725. doi: 10.1038/s41467-022-30008-0.
  PUBMED    35585055
REFERENCE   2  (bases 1 to 8168)
  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 
            Commun."; date: "2022-05-18"; volume: "13(1):2725. doi"; pages: " 
            10.1038/s41467-022-30008-0"
COMMENT     Sequence Label: EYFP NES visual barcode
FEATURES             Location/Qualifiers
     source          1..8168
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     complement(534..553)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1030..1050
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1170..1235
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             join(1242..1244,1245..1247,1248..1955)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(1287..1308)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1548..1567)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1895..1916
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2024..2612
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2077..2097)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2495..2506)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2684..2917
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2995..3116
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3032..3051)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3086..3105
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3156..3291
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3311..3330)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3312..3330)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3340..3357)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3340..3356)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3349..3371)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3498..3953
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3585..3604)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3795..3816
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3979..4083
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4084..4152,4153..4944)
                     /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(4302..4321)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5115..5703
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5604..5623
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5857..5874
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5991..6012
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6027..6032,6033..6050,6051..6057)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6065..6081
                     /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     6070..6092
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6089..6105
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6089..6105
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6124..6144
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6126..6144
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6172..6398
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6399..6579
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6626..6751
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7244..7477
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7662..7706
                     /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    7935..8052
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctct ctggctaact gtcgggatcc accatgaccg
      541 agtacaagcc caccgtgcgc ctcgccaccc gcgacgacgt gcccagggcc gtccgcaccc
      601 tcgccgccgc tttcgccgac taccccgcca cccgccacac cgtcgatccc gaccgccaca
      661 tcgagcgggt caccgagctg caagaactct tcctcacccg cgtcgggctc gacatcggca
      721 aggtgtgggt cgctgacgac ggcgccgcag tggctgtctg gaccacaccc gagagcgtcg
      781 aagctggggc ggtgttcgcc gagatcggcc ctcgcatggc cgagttgagc ggttcccggc
      841 tggccgctca gcaacagatg gaaggcctcc tggctccaca ccggcccaag gagcccgcat
      901 ggttcctggc caccgtcggc gtctctcccg accaccaggg caagggtctg ggcagcgccg
      961 tcgtgctccc cggagtggag gctgccgagc gcgccggggt gcccgccttc ctggagacct
     1021 ccgcaccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtgg
     1081 aggtgcccga aggaccacgc acctggtgca tgacccgcaa gcccggtgcc ctgcagggat
     1141 ctggcgaagc cagacacaaa cagaaaattg tgaaacagac tttgaatttt gaccttctca
     1201 agttggcggg agacgtggag tccaacccag ggcccgctag catggtgagc aagggcgagg
     1261 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1321 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     1381 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcggct
     1441 acggcctgca gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1501 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     1561 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1621 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     1681 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     1741 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     1801 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc taccagtccg
     1861 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     1921 ccgccgggat cactctcggc atggacgagc tgtacagtga gctgcagaac aagctggaag
     1981 agttggatct ggactcgtac aagtaaagac gcgttaagtc gacaatcaac ctctggatta
     2041 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     2101 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     2161 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     2221 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     2281 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     2341 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     2401 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     2461 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     2521 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     2581 gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag
     2641 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     2701 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
     2761 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     2821 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     2881 ctcagaccct tttagtcagt gtggaaaatc tctagcagta cgtatagtag ttcatgtcat
     2941 cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg
     3001 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3061 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     3121 gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3181 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3241 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt
     3301 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     3361 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     3421 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     3481 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3541 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3601 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3661 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3721 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3781 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3841 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3901 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     3961 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4021 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4081 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4141 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4201 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4261 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4321 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4381 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4441 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4501 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4561 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4621 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4681 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4741 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4801 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4861 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4921 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4981 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     5041 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5101 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5161 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5221 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     5281 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5341 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5401 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5461 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5521 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5581 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5641 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5701 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5761 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5821 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5881 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     5941 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     6001 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     6061 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     6121 cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt
     6181 atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag
     6241 gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat
     6301 taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca
     6361 gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga
     6421 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     6481 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     6541 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
     6601 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
     6661 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
     6721 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
     6781 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
     6841 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
     6901 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     6961 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     7021 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     7081 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     7141 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     7201 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     7261 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     7321 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     7381 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     7441 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     7501 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     7561 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     7621 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     7681 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     7741 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     7801 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc
     7861 tcccaacccc gaggggaccc gatatcgcgg ccgcaattct agatcttgag acaaatggca
     7921 gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga
     7981 atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa
     8041 attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcg ccggctcgag
     8101 ggggcccggg gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     8161 gagttccg
//
