LOCUS       sequence-309764-        8090 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid serves as the backbone for the visual barcodes 
            described in our paper. It has a CMV-Puro-F2A backbone to which we 
            insert a fluorescent protein cellular localization combination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8090)
  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 8090)
  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: Barcode backbone
FEATURES             Location/Qualifiers
     source          1..8090
                     /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             1242..1928
                     /codon_start=1
                     /product="far-red fluorescent variant of Heteractis crispa 
                     chromoprotein"
                     /label=HcRed1
                     /note="mammalian codon-optimized"
                     /translation="MVSGLLKESMRIKMYMEGTVNGHYFKCEGEGDGNPFAGTQSMRIH
                     VTEGAPLPFAFDILAPCCEYGSRTFVHHTAEIPDFFKQSFPEGFTWERTTTYEDGGILT
                     AHQDTSLEGNCLIYKVKVHGTNFPADGPVMKNKSGGWEPSTEVVYPENGVLCGRNVMAL
                     KVGDRHLICHHYTSYRSKKAVRALTMPGFHFTDIRLQMLRKKKDEYFELYEASVARYSD
                     LPEKAN"
     misc_feature    1946..2534
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1999..2019)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2417..2428)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2606..2839
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2917..3038
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2954..2973)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3008..3027
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3078..3213
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3233..3252)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3234..3252)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3262..3279)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3262..3278)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3271..3293)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3420..3875
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3507..3526)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3717..3738
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3901..4005
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4006..4074,4075..4866)
                     /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(4224..4243)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5037..5625
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5526..5545
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5779..5796
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5913..5934
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5949..5954,5955..5972,5973..5979)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5987..6003
                     /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     5992..6014
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6011..6027
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6011..6027
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6046..6066
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6048..6066
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6094..6320
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6321..6501
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6548..6673
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7166..7399
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7584..7628
                     /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    7857..7974
                     /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 ggcctgctga
     1261 aggagagtat gcgcatcaag atgtacatgg agggcaccgt gaacggccac tacttcaagt
     1321 gcgagggcga gggcgacggc aaccccttcg ccggcaccca gagcatgaga atccacgtga
     1381 ccgagggcgc ccccctgccc ttcgccttcg acatcctggc cccctgctgc gagtacggca
     1441 gcaggacctt cgtgcaccac accgccgaga tccccgactt cttcaagcag agcttccccg
     1501 agggcttcac ctgggagaga accaccacct acgaggacgg cggcatcctg accgcccacc
     1561 aggacaccag cctggagggc aactgcctga tctacaaggt gaaggtgcac ggcaccaact
     1621 tccccgccga cggccccgtg atgaagaaca agagcggcgg ctgggagccc agcaccgagg
     1681 tggtgtaccc cgagaacggc gtgctgtgcg gccggaacgt gatggccctg aaggtgggcg
     1741 accggcacct gatctgccac cactacacca gctaccggag caagaaggcc gtgcgcgccc
     1801 tgaccatgcc cggcttccac ttcaccgaca tccggctcca gatgctgcgg aagaagaagg
     1861 acgagtactt cgagctgtac gaggccagcg tggcccggta cagcgacctg cccgagaagg
     1921 ccaactgaag acgcgttaag tcgacaatca acctctggat tacaaaattt gtgaaagatt
     1981 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     2041 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     2101 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     2161 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     2221 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     2281 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     2341 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     2401 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     2461 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     2521 ggccgcctcc ccgcgtcgac tttaagacca atgacttaca aggcagctgt agatcttagc
     2581 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     2641 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     2701 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     2761 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     2821 gtgtggaaaa tctctagcag tacgtatagt agttcatgtc atcttattat tcagtattta
     2881 taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat
     2941 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     3001 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc
     3061 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3121 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     3181 tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag
     3241 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     3301 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     3361 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     3421 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3481 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3541 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3601 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3661 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3721 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3781 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3841 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     3901 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3961 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     4021 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     4081 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     4141 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     4201 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     4261 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     4321 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     4381 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     4441 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     4501 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     4561 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     4621 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     4681 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     4741 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     4801 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     4861 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     4921 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4981 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5041 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5101 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5161 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     5221 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5281 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5341 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5401 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5461 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5521 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5581 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5641 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5701 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     5761 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     5821 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     5881 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     5941 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     6001 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
     6061 taaagggaac aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt
     6121 cttgcaacat ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg
     6181 catgccgatt ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg
     6241 gtctgacatg gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt
     6301 gcctagctcg atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct
     6361 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     6421 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     6481 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
     6541 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
     6601 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
     6661 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
     6721 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
     6781 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
     6841 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     6901 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     6961 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga
     7021 tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata
     7081 aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc
     7141 agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag
     7201 gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg
     7261 gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc
     7321 aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc
     7381 taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg
     7441 ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga
     7501 cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg
     7561 aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg
     7621 caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa
     7681 tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata
     7741 gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac
     7801 ccgatatcgc ggccgcaatt ctagatcttg agacaaatgg cagtattcat ccacaatttt
     7861 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     7921 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     7981 tattacaggg acagcagaga tccactttgg cgccggctcg agggggcccg gggttattaa
     8041 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
//
