LOCUS       sequence-462234-        8089 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 mScarlet marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8089)
  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 8089)
  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-mScarlet
FEATURES             Location/Qualifiers
     source          1..8089
                     /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"
     regulatory      3106..3115
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3112..3804
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELY"
     primer_bind     complement(3245..3263)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3513..3532
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(3841..3865)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3883..4471
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3936..3956)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4354..4365)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4610..4843
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4915..5036
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4952..4971)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5006..5025
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5076..5211
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5231..5250)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5232..5250)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5260..5277)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5260..5276)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5269..5291)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5418..5873
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5505..5524)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5715..5736
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5899..6003
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6004..6072,6073..6864)
                     /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(6222..6241)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7035..7623
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7524..7543
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7777..7794
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7911..7932
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7947..7952,7953..7970,7971..7977)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7985..8001
                     /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     7990..8012
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8009..8025
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8009..8025
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8044..8064
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8046..8064
                     /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 catggtgagc
     3121 aagggcgagg cagtgatcaa ggagttcatg cggttcaagg tgcacatgga gggctccatg
     3181 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     3241 accgccaagc tgaaggtgac caagggtggc cccctgccct tctcctggga catcctgtcc
     3301 cctcagttca tgtacggctc cagggccttc accaagcacc ccgccgacat ccccgactac
     3361 tataagcagt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     3421 ggcgccgtga ccgtgaccca ggacacctcc ctggaggacg gcaccctgat ctacaaggtg
     3481 aagctccgcg gcaccaactt ccctcctgac ggccccgtaa tgcagaagaa gacaatgggc
     3541 tgggaagcgt ccaccgagcg gttgtacccc gaggacggcg tgctgaaggg cgacattaag
     3601 atggccctgc gcctgaagga cggcggccgc tacctggcgg acttcaagac cacctacaag
     3661 gccaagaagc ccgtgcagat gcccggcgcc tacaacgtcg accgcaagtt ggacatcacc
     3721 tcccacaacg aggactacac cgtggtggaa cagtacgaac gctccgaggg ccgccactcc
     3781 accggcggca tggacgagct gtactaatag ctcgagcccg ggtctagagt cgaccatatg
     3841 acccagcttt cttgtacaaa gtggtgataa tcgaattccg ataatcaacc tctggattac
     3901 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     3961 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     4021 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     4081 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     4141 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     4201 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     4261 gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg
     4321 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     4381 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     4441 agtcggatct ccctttgggc cgcctccccg catcggttgc taggagagac catatgggat
     4501 ccgatatcgc caccgtggct gaatgagact ggcccgcggt tcgctttaag accaatgact
     4561 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     4621 attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac
     4681 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     4741 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     4801 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat
     4861 cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg
     4921 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4981 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     5041 gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     5101 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     5161 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt
     5221 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     5281 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     5341 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     5401 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5461 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     5521 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5581 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     5641 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5701 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     5761 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     5821 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     5881 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5941 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     6001 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     6061 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     6121 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     6181 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     6241 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     6301 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     6361 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     6421 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6481 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6541 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6601 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6661 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6721 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6781 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6841 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6901 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6961 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     7021 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     7081 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     7141 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     7201 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     7261 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     7321 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     7381 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     7441 acgcttcccg aagagagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     7501 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7561 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7621 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     7681 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     7741 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     7801 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     7861 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     7921 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     7981 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     8041 cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctt
//
