LOCUS       sequence-309995-        7459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of sgRNA against human CD19.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7459)
  TITLE     Lenitviral sgRNA and Cas9 constructs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7459)
  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: 
            "Unpublished"
COMMENT     Sequence Label: Lentiviral sgRNA human CD19
FEATURES             Location/Qualifiers
     source          1..7459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     134..154
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        136..154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        182..408
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             409..589
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    636..761
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1254..1487
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1672..1716
                     /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"
     promoter        1894..2142
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1894..1914
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2065..2084
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2308..2425
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2494..2705
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2725..2734
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2734..2757
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(2758..2760,2761..2763,2764..3477)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2758..2760,2761..2763,2764..3474)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2803..2824)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3064..3083)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3411..3432
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3493..4081
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3546..3566)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3964..3975)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4153..4386
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4464..4585
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4501..4520)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4555..4574
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4625..4760
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4780..4799)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4781..4799)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4809..4826)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4809..4825)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4818..4840)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4967..5422
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5054..5073)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5264..5285
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5448..5552
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5553..5621,5622..6413)
                     /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(5771..5790)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6584..7172
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7073..7092
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7326..7343
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta
      181 atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat
      241 gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat
      301 cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg
      361 ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg
      421 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      481 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      541 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
      601 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
      661 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
      721 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
      781 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
      841 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
      901 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
      961 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1021 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1081 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1141 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1201 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1261 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1321 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1381 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1441 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1501 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1561 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     1621 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     1681 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     1741 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     1801 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     1861 cagacccacc tcccaacccc gaggggaccc agagagggcc tatttcccat gattccttca
     1921 tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac
     1981 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     2041 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     2101 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgaagcggg gactcccgag
     2161 accgtttaag agctatgctg gaaacagcat agcaagttta aataaggcta gtccgttatc
     2221 aactttgctg gaaacagcaa agtggcaccg agtcggtgct ttttttgaat tctagatctt
     2281 gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     2341 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     2401 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg
     2461 gcgccggctc gagtggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
     2521 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
     2581 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
     2641 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
     2701 cacaggtgtc gtgacgcggg atccgccacc atggattaca aagacgatga cgataagatg
     2761 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     2821 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     2881 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     2941 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     3001 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     3061 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     3121 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     3181 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     3241 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     3301 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     3361 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     3421 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtagacg
     3481 cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     3541 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     3601 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     3661 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     3721 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     3781 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     3841 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     3901 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     3961 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     4021 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     4081 cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     4141 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
     4201 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     4261 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     4321 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     4381 ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga
     4441 aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa
     4501 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4561 tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc
     4621 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4681 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     4741 aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac
     4801 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     4861 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     4921 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     4981 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     5041 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     5101 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5161 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     5221 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5281 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     5341 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5401 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5461 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5521 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5581 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5641 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5701 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5761 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5821 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5881 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5941 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     6001 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6061 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6121 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6181 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6241 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6301 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6361 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6421 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6481 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6541 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6601 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6661 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6721 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6781 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6841 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6901 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6961 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     7021 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     7081 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7141 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7201 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     7261 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     7321 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     7381 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     7441 gcagtgagcg caacgcaat
//
