LOCUS       sequence-462163-        8110 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning expressed barcodes at the EcoRV site for preparing 
            lentiviral libaries for single-cell lineage tracing experiments, 
            with T-Sapphire marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8110)
  AUTHORS   Singh I, Fernandez-Perez D, Sanchez PS, Rodriguez-Fraticelli AE
  TITLE     Pre-existing stem cell heterogeneity dictates clonal responses to 
            the acquisition of leukemic driver mutations.
  JOURNAL   Cell Stem Cell. 2025 Feb 19:S1934-5909(25)00012-8. doi: 
            10.1016/j.stem.2025.01.012.
  PUBMED    40010350
REFERENCE   2  (bases 1 to 8110)
  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: "Cell Stem 
            Cell. 2025 Feb 19:S1934-5909(25)00012-8. doi: 
            10.1016/j.stem.2025.01.012."
COMMENT     Sequence Label: pLARRYv2-T-Sapphire
FEATURES             Location/Qualifiers
     source          1..8110
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..229
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             230..410
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    457..582
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1075..1308
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1493..1537
                     /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    1803..1920
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1927..3105
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2158..3096
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3053..3073
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3112..3828
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVMVFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSIQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3142..3170)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3765..3785
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    complement(3862..3886)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3904..4492
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3957..3977)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4375..4386)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4631..4864
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4936..5057
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4973..4992)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5027..5046
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5097..5232
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5252..5271)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5253..5271)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5281..5298)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5281..5297)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5290..5312)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5439..5894
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5526..5545)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5736..5757
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5920..6024
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6025..6093,6094..6885)
                     /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(6243..6262)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7056..7644
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7545..7564
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7798..7815
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7932..7953
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7968..7973,7974..7991,7992..7998)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8006..8022
                     /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     8011..8033
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8030..8046
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8030..8046
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8065..8085
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8067..8085
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca
       61 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
      121 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt
      181 gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
      241 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
      301 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
      361 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
      421 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
      481 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg
      541 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
      601 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta
      661 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
      721 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
      781 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
      841 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt
      901 aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
      961 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
     1021 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
     1081 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
     1141 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
     1201 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
     1261 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
     1321 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
     1381 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
     1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgcta
     1801 gcttttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
     1861 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
     1921 actagtggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     1981 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
     2041 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     2101 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta
     2161 agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct
     2221 tgaattactt ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt
     2281 gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag
     2341 gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc
     2401 gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt
     2461 ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt
     2521 ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc
     2581 ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg
     2641 gtgcctggtc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg
     2701 gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa
     2761 tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc
     2821 tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac
     2881 ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat
     2941 gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg
     3001 atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct
     3061 cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgagccac catgagtaaa
     3121 ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat
     3181 gggcacaaat tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc
     3241 cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact tgtcactact
     3301 ttctcttatg gtgttatggt ctttgcgaga tacccagatc atatgaaaca gcatgacttt
     3361 ttcaagagtg ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac
     3421 gggaactaca agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc
     3481 gagttaaaag gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac
     3541 aactttaact cacacaatgt atacatcatg gcagacaaac aaaagaatgg aatcaaagcg
     3601 aacttcaaaa ttagacacaa cattgaagat ggaggcgttc aactagcaga ccattatcaa
     3661 caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta cctgtccata
     3721 caatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt
     3781 gtaacagctg ctgggattac acatggcatg gatgaactat acaaataata gctcgagccc
     3841 gggtctagag tcgaccatat gacccagctt tcttgtacaa agtggtgata atcgaattcc
     3901 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     3961 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     4021 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     4081 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     4141 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     4201 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     4261 ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt tccatggctg
     4321 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     4381 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     4441 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcggttg
     4501 ctaggagaga ccatatggga tccgatatcg ccaccgtggc tgaatgagac tggcccgcgg
     4561 ttcgctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     4621 aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt
     4681 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     4741 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     4801 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     4861 agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     4921 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4981 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5041 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc
     5101 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     5161 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     5221 tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     5281 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     5341 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     5401 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     5461 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     5521 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5581 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5641 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5701 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5761 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     5821 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5881 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5941 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     6001 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     6061 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     6121 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6181 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6241 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6301 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6361 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6421 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6481 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6541 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6601 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6661 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6721 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6781 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6841 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6901 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6961 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     7021 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     7081 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     7141 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     7201 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     7261 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     7321 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     7381 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     7441 cgtgagctat gagaaagcgc cacgcttccc gaagagagaa aggcggacag gtatccggta
     7501 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7561 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7621 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7681 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     7741 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     7801 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     7861 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     7921 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     7981 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     8041 tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag
     8101 ctgcaagctt
//
