LOCUS       sequence-266186-        7480 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector for sgRNA expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7480)
  AUTHORS   Huang Y, Marovca B, Dettwiler S, Bode PK, Bornhauser B, Bourquin JP
  TITLE     Rapid Generation of Leukemogenic Chromosomal Translocations in Vivo 
            Using CRISPR/Cas9.
  JOURNAL   Hemasphere. 2020 Sep 30;4(5):e456. doi: 
            10.1097/HS9.0000000000000456. eCollection 2020 Oct.
  PUBMED    33134860
REFERENCE   2  (bases 1 to 7480)
  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: 
            "Hemasphere."; date: "2020-09-30"; volume: "4(5):e456. doi"; pages: 
            " 10.1097/HS9.0000000000000456. eCollection 2020 Oct"
COMMENT     Sequence Label: sg_shuttle_RFP657
FEATURES             Location/Qualifiers
     source          1..7480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2234..2482
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2234..2254
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2405..2424
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2501..2576
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2689..2900
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2926..3627
                     /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    3649..4237
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3702..3722)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4120..4131)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4309..4542
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(4568..4585)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4574..4798
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4885..4903)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4971..5075
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5076..5144,5145..5936)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5294..5313)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6107..6695
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6596..6615
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6849..6866
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7106..7127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7142..7147,7148..7165,7166..7172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7180..7196
                     /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     7185..7207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7204..7220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7204..7220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7270..7289
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactaggatc
     2221 cactagtaca tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     2281 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     2341 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     2401 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     2461 cttgtggaaa ggacgaaaca ccggagacgc gagcgtctct gttttagagc tagaaatagc
     2521 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     2581 ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc
     2641 caattctgca gacaaatggc tctagaggta cctccggtgc ccgtcagtgg gcagagcgca
     2701 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
     2761 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
     2821 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     2881 ggtttgccgc cagaacacag gtgtcgtgac gcgggatctg ccaccatgag cgagctgatc
     2941 accgagaaca tgcacatgaa gctgtacatg gagggcaccg tgaacaacca ccacttcaag
     3001 tgcacatccg agggcgaagg caagccctac gagggcaccc agacccagag aatcaaggtg
     3061 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctaccagctt catgtacggc
     3121 agtcacacct tcatcaacca cacccagggc atccccgact tctggaagca gtccttccct
     3181 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
     3241 caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac
     3301 ttcccatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc ccacaccgag
     3361 atgctgtacc ccgctgacgg cggcctggaa ggcagaaccg cgctggccct gaagctcgtg
     3421 ggcgggggcc acctgatctg caacttcaag accacataca gatccaagaa acccgctaag
     3481 aacctcaaga tgcccggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc
     3541 gacaaagaga cctacgtcga gcagcacgag gtggctgtgg ccagatactg cgacctccct
     3601 agcaaactgg ggcacaagct taattaaggt accacgcgtt aagtcgacaa tcaacctctg
     3661 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     3721 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     3781 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     3841 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     3901 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     3961 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     4021 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     4081 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     4141 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     4201 cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt
     4261 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     4321 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     4381 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     4441 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     4501 gatccctcag acccttttag tcagtgtgga aaatctctag cagggcccgt ttaaacccgc
     4561 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     4621 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     4681 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     4741 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggtt
     4801 ataacttgca aagaaatgaa tatcagagag tgagaggccc gggttaatta aggaaagggc
     4861 tagatcattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     4921 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     4981 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5041 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5101 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5161 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5221 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5281 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     5341 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5401 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5461 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5521 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     5581 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     5641 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     5701 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     5761 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     5821 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     5881 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     5941 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6001 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6061 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6121 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6181 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6241 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6301 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6361 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6421 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6481 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6541 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6601 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6661 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6721 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     6781 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     6841 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     6901 tccccgcgcg ttggccgatt cattaatgca gcaagctcat ggctgactaa ttttttttat
     6961 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     7021 ttttggaggc ctaggctttt gcaaaaagct ccccgtggca cgacaggttt cccgactgga
     7081 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     7141 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     7201 acacaggaaa cagctatgac atgattacga atttcacaaa taaagcattt ttttcactgc
     7261 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcaactgga
     7321 taactcaagc taaccaaaat catcccaaac ttcccacccc ataccctatt accactgcca
     7381 attacctgtg gtttcattta ctctaaacct gtgattcctc tgaattattt tcattttaaa
     7441 gaaattgtat ttgttaaata tgtactacaa acttagtagt
//
