LOCUS       sequence-189853-        8181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Advanced lentiviral Vector for sgRNA (hU6 Promoter) delivery with 
            RFP657 expression (EFS Promoter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8181)
  AUTHORS   Reimer J, Knoess S, Labuhn M, Charpentier EM, Gohring G, 
            Schlegelberger B, Klusmann JH, Heckl D
  TITLE     CRISPR-Cas9-induced t(11;19)/MLL-ENL translocations initiate 
            leukemia in human hematopoietic progenitor cells in vivo.
  JOURNAL   Haematologica. 2017 Jun 1. pii: haematol.2017.164046. doi: 
            10.3324/haematol.2017.164046.
  PUBMED    28572162
REFERENCE   2  (bases 1 to 8181)
  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: 
            "Haematologica. 2017 Jun 1. pii: haematol.2017.164046. doi: 
            10.3324/haematol.2017.164046."
COMMENT     Sequence Label: SGL40C.EFS.RFP657
FEATURES             Location/Qualifiers
     source          1..8181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     101..118
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    235..256
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(271..276,277..294,295..301)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    309..325
                     /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     314..336
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     333..349
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     333..349
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     368..388
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        370..388
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     enhancer        433..812
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     enhancer        869..1060
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(892..912)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1079..1305
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1306..1486
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1533..1658
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2151..2384
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2569..2613
                     /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    2911..3028
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3079..3327
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3079..3099
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3250..3269
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3489..3700
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             3726..4427
                     /codon_start=1
                     /product="far-red monomeric fluorescent protein with an 
                     emission peak at 657 nm (Morozova et al., 2010)"
                     /label=TagRFP657
                     /note="derived from mKate"
                     /translation="MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTQRIKV
                     VEGGPLPFAFDILATSFMYGSHTFINHTQGIPDFWKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEAHTEMLYPADGGLEGRTALALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDYRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    4449..5037
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4502..4522)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4920..4931)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5109..5342
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5420..5541
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5457..5476)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5511..5530
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5581..5716
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5736..5755)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5737..5755)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5765..5782)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5765..5781)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5774..5796)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5923..6378
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6010..6029)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6220..6241
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6404..6508
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6509..6577,6578..7369)
                     /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(6727..6746)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7540..8128
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8029..8048
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
       61 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      121 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      181 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      241 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      301 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      361 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag cggtaccgtc
      421 gacctgcagg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      481 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      541 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      601 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      661 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      721 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      781 acatctacgt attagtcatc gctattacca tggcgcgcca tgcagggcct gaaataacct
      841 ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt
      901 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
      961 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     1021 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc ttaagaggcg cgccttaatg
     1081 tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc
     1141 ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt
     1201 gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg
     1261 cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta
     1321 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     1381 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     1441 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     1501 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     1561 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     1621 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     1681 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     1741 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     1801 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     1861 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     1921 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     1981 ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat
     2041 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     2101 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     2161 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     2221 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     2281 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     2341 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     2401 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     2461 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     2521 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     2581 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     2641 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     2701 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     2761 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     2821 gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg tgcgccaatt
     2881 ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     2941 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     3001 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt
     3061 ttggttaatt aaggtaccga gggcctattt cccatgattc cttcatattt gcatatacga
     3121 tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta
     3181 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     3241 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     3301 tatatcttgt ggaaaggacg aaacaccgga gacgtatgtc gacaactcgt ctctgtttaa
     3361 gagctatgct ggaaacagca tagcaagttt aaataaggct agtccgttat caacttgaaa
     3421 aagtggcacc gagtcggtgc ttttttgaat tctcctctag aactcgagtg gctccggtgc
     3481 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     3541 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     3601 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     3661 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac gcgggatctg
     3721 ccaccatgag cgagctgatc accgagaaca tgcacatgaa gctgtacatg gagggcaccg
     3781 tgaacaacca ccacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc
     3841 agacccagag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg
     3901 ctaccagctt catgtacggc agtcacacct tcatcaacca cacccagggc atccccgact
     3961 tctggaagca gtccttccct gagggcttca catgggagag agtcaccaca tacgaagacg
     4021 ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg
     4081 tcaagatcag aggggtgaac ttcccatcca acggccctgt gatgcagaag aaaacactcg
     4141 gctgggaggc ccacaccgag atgctgtacc ccgctgacgg cggcctggaa ggcagaaccg
     4201 cgctggccct gaagctcgtg ggcgggggcc acctgatctg caacttcaag accacataca
     4261 gatccaagaa acccgctaag aacctcaaga tgcccggcgt ctactatgtg gactacagac
     4321 tggaaagaat caaggaggcc gacaaagaga cctacgtcga gcagcacgag gtggctgtgg
     4381 ccagatactg cgacctccct agcaaactgg ggcacaagct taattaaggt accacgcgtt
     4441 aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     4501 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     4561 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     4621 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     4681 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     4741 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     4801 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     4861 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     4921 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     4981 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc
     5041 gactttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa
     5101 ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac
     5161 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     5221 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     5281 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     5341 cagtacgtat agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg
     5401 aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa
     5461 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5521 caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc
     5581 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     5641 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     5701 ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc
     5761 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     5821 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     5881 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     5941 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     6001 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     6061 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     6121 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     6181 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     6241 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     6301 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     6361 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     6421 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     6481 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     6541 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     6601 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     6661 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     6721 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     6781 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6841 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6901 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6961 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     7021 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     7081 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     7141 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     7201 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     7261 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     7321 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     7381 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     7441 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     7501 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     7561 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     7621 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     7681 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     7741 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7801 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7861 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7921 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7981 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     8041 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     8101 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     8161 ggccttttgc tggccttttg c
//
