LOCUS       sequence-311031-        8107 bp    DNA     circular SYN 14-APR-2025
DEFINITION  tagging clonal populations with whole cell acGFP fluorescent 
            protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8107)
  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 8107)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: acGFP WC visual barcode
FEATURES             Location/Qualifiers
     source          1..8107
                     /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..1961
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     misc_feature    1979..2567
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2032..2052)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2450..2461)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2639..2872
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2950..3071
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2987..3006)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3041..3060
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3111..3246
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3266..3285)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3267..3285)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3295..3312)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3295..3311)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3304..3326)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3453..3908
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3540..3559)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3750..3771
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3934..4038
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4039..4107,4108..4899)
                     /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(4257..4276)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5070..5658
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5559..5578
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5812..5829
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5946..5967
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5982..5987,5988..6005,6006..6012)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6020..6036
                     /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     6025..6047
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6044..6060
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6044..6060
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6079..6099
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6081..6099
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6127..6353
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6354..6534
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6581..6706
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7199..7432
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7617..7661
                     /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    7874..7991
                     /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 aagggcgccg
     1261 agctgttcac cggcatcgtg cccatcctga tcgagctgaa tggcgatgtg aatggccaca
     1321 agttcagcgt gagcggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     1381 tcatctgcac caccggcaag ctgcctgtgc cctggcccac cctggtgacc accctgagct
     1441 acggcgtgca gtgcttctca cgctaccccg atcacatgaa gcagcacgac ttcttcaaga
     1501 gcgccatgcc tgagggctac atccaggagc gcaccatctt cttcgaggat gacggcaact
     1561 acaagtcgcg cgccgaggtg aagttcgagg gcgataccct ggtgaatcgc atcgagctga
     1621 ccggcaccga tttcaaggag gatggcaaca tcctgggcaa taagatggag tacaactaca
     1681 acgcccacaa tgtgtacatc atgaccgaca aggccaagaa tggcatcaag gtgaacttca
     1741 agatccgcca caacatcgag gatggcagcg tgcagctggc cgaccactac cagcagaata
     1801 cccccatcgg cgatggccct gtgctgctgc ccgataacca ctacctgtcc acccagagcg
     1861 ccctgtccaa ggaccccaac gagaagcgcg atcacatgat ctacttcggc ttcgtgaccg
     1921 ccgccgccat cacccacggc atggatgagc tgtacaagtg aagacgcgtt aagtcgacaa
     1981 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     2041 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     2101 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     2161 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     2221 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     2281 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     2341 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     2401 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     2461 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     2521 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga
     2581 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     2641 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     2701 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     2761 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     2821 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat
     2881 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
     2941 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3001 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3061 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc
     3121 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     3181 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     3241 gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     3301 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     3361 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     3421 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     3481 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3541 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3601 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3661 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3721 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3781 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3841 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     3901 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3961 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     4021 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     4081 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     4141 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     4201 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     4261 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     4321 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     4381 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     4441 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     4501 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     4561 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     4621 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     4681 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     4741 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     4801 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     4861 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     4921 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     4981 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5041 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5101 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5161 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     5221 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     5281 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     5341 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     5401 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     5461 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     5521 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     5581 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     5641 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     5701 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     5761 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     5821 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     5881 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     5941 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     6001 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     6061 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa
     6121 gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc
     6181 aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt
     6241 acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg
     6301 aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc
     6361 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     6421 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     6481 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
     6541 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     6601 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     6661 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     6721 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     6781 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     6841 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     6901 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     6961 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     7021 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga
     7081 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag
     7141 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag
     7201 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga
     7261 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc
     7321 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc
     7381 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt
     7441 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta
     7501 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta
     7561 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga
     7621 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa
     7681 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa
     7741 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat
     7801 cgtttcagac ccacctccca accccgaggg gacaattcta gatcttgaga caaatggcag
     7861 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     7921 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     7981 ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggcgc cggctcgagg
     8041 gggcccgggg ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     8101 agttccg
//
