LOCUS       sequence-349887-        8249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Neomycin-targeting gRNA (mU6), a non-targeting gRNA 
            (hU6) and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8249)
  TITLE     Winslow lab 9-24-2021 deposit
REFERENCE   2  (bases 1 to 8249)
  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"
COMMENT     Sequence Label: mU6-sgNeo1-hU6-sgNT
FEATURES             Location/Qualifiers
     source          1..8249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             162..342
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(404..992)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(484..503)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1163..1954,1955..2023))
                     /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     1786..1805
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2024..2128)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2203..2222)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2394..2773
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2775..2973
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2928..2948
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2991..3171
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3218..3343
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3840..4073
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4258..4302
                     /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    4600..4717
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     5030..5050
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     5276..5296
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5447..5466
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        5686..6185
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5721..5744)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6118..6137
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6189..6209
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6210..7238
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     misc_feature    7294..7882
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7347..7367)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7765..7776)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7885..7901)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7886..7902)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 aagagaaggt agaagaagcc aatgaaggag agaacacccg cttgttacac cctgtgagcc
       61 tgcatgggat ggatgacccg gagagagaag tattagagtg gaggtttgac agccgcctag
      121 catttcatca catggcccga gagctgcatc cggactgtac tgggtctctc tggttagacc
      181 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      241 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
      301 gatccctcag acccttttag tcagtgtgga aaatctctag cagcatgtga gcaaaaggcc
      361 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
      421 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
      481 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
      541 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
      601 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
      661 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
      721 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
      781 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
      841 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
      901 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
      961 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     1021 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     1081 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     1141 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     1201 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     1261 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     1321 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     1381 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     1441 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     1501 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     1561 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     1621 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     1681 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     1741 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     1801 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     1861 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     1921 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     1981 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     2041 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     2101 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     2161 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     2221 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     2281 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     2341 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
     2401 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     2461 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     2521 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     2581 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     2641 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     2701 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     2761 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     2821 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     2881 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     2941 taggcgtgta cggtgggagg tctatataag cagcgcgttt tgcctgtact gggtctctct
     3001 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     3061 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     3121 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
     3181 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
     3241 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
     3301 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
     3361 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
     3421 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
     3481 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
     3541 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
     3601 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
     3661 taagaccacc gcacagcaag cggccggccg ctgatcttca gacctggagg aggagatatg
     3721 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     3781 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     3841 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     3901 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     3961 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     4021 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     4081 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     4141 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     4201 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     4261 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     4321 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     4381 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     4441 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     4501 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     4561 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     4621 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     4681 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     4741 agatccagtt tggttagtac cgggcccgct ctagccctgc aggaactgtt taaactgtga
     4801 tccgacgcgc catctctagg cccgcgccgg ccccctcgca cggacttgtg ggagaagctc
     4861 ggctactccc ctgccccggt taatttgcat ataatatttc ctagtaacta tagaggctta
     4921 atgtgcgata aaagacagat aatctgttct ttttaatact agctacattt tacatgatag
     4981 gcttggattt ctataagaga tacaaatact aaattattat tttaaaaaac agcacaaaag
     5041 gaaactcacc ctaactgtaa agtaattgtg tgttttgaga ctataagtat cccttggaga
     5101 accaccttgt tggtcatggc tgatgcaatg cgggtttgag agctaagcag aaagctgcat
     5161 agcaagttca aataaggcta gtccgtacac aacttgaaaa agtggcagcc gagtcggctg
     5221 ctttttttgc tcgaatctac actcagctat ggcgcgcccc aaggtcgggc aggaagaggg
     5281 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     5341 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     5401 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
     5461 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aagccagaaa
     5521 catggcgagg tattcggctc cgcggtttca gagctaagca caagagtgca tagcaagttg
     5581 aaataaggct agtccgttta caacttgaaa aagtggcacc cgagtcgggt gctttttttg
     5641 tttggcgcgc ctatggctta attaacatgg cggccgcatt ctaccgggta ggggaggcgc
     5701 ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca
     5761 caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt
     5821 tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg
     5881 ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg
     5941 tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
     6001 agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg
     6061 ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat
     6121 tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggac
     6181 ccgggatgcc caagaagaag aggaaggtgt ccaatttact gaccgtacac caaaatttgc
     6241 ctgcattacc ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg gacatgttca
     6301 gggatcgcca ggcgttttct gagcatacct ggaaaatgct tctgtccgtt tgccggtcgt
     6361 gggcggcatg gtgcaagttg aataaccgga aatggtttcc cgcagaacct gaagatgttc
     6421 gcgattatct tctatatctt caggcgcgcg gtctggcagt aaaaactatc cagcaacatt
     6481 tgggccagct aaacatgctt catcgtcggt ccgggctgcc acgaccaagt gacagcaatg
     6541 ctgtttcact ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt gaacgtgcaa
     6601 aacaggctct agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc atggaaaata
     6661 gcgatcgctg ccaggatata cgtaatctgg catttctggg gattgcttat aacaccctgt
     6721 tacgtatagc cgaaattgcc aggatcaggg ttaaagatat ctcacgtact gacggtggga
     6781 gaatgttaat ccatattggc agaacgaaaa cgctggttag caccgcaggt gtagagaagg
     6841 cacttagcct gggggtaact aaactggtcg agcgatggat ttccgtctct ggtgtagctg
     6901 atgatccgaa taactacctg ttttgccggg tcagaaaaaa tggtgttgcc gcgccatctg
     6961 ccaccagcca gctatcaact cgcgccctgg aagggatttt tgaagcaact catcgattga
     7021 tttacggcgc taaggatgac tctggtcaga gatacctggc ctggtctgga cacagtgccc
     7081 gtgtcggagc cgcgcgagat atggcccgcg ctggagtttc aataccggag atcatgcaag
     7141 ctggtggctg gaccaatgta aatattgtca tgaactatat ccgtaacctg gatagtgaaa
     7201 caggggcaat ggtgcgcctg ctggaagatg gcgattagcc atgtttaagg gttccggttc
     7261 cactaggtac aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt
     7321 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     7381 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     7441 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     7501 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     7561 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     7621 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     7681 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
     7741 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     7801 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     7861 tccctttggg ccgcctcccc gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca
     7921 tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc
     7981 acaagaggag gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac
     8041 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     8101 aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta
     8161 cttccctgat tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg
     8221 atggtgctac aagctagtac cagttgagc
//
