LOCUS       sequence-352695-        8673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Neomycin (bU6), Nuak1 (mU6) and Nuak2(hU6) gRNAs and 
            Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8673)
  TITLE     Winslow lab 9-24-2021 deposit
REFERENCE   2  (bases 1 to 8673)
  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: bU6-sgNeo1-mU6-sgNuak1-hU6-sgNuak2
FEATURES             Location/Qualifiers
     source          1..8673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             69..249
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(311..899)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(391..410)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1070..1861,1862..1930))
                     /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     1693..1712
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1931..2035)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2110..2129)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2301..2680
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2682..2880
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2835..2855
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2898..3078
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3125..3250
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3747..3980
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4165..4209
                     /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    4507..4624
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     5361..5381
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     5607..5627
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5778..5797
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        6017..6516
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6052..6075)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6449..6468
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6520..6540
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6541..7569
                     /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    7625..8213
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7678..7698)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8096..8107)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8216..8232)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8217..8233)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg
       61 actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
      121 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
      181 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
      241 tctctagcag catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
      301 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
      361 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
      421 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
      481 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
      541 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
      601 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
      661 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
      721 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
      781 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
      841 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
      901 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
      961 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     1021 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     1081 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     1141 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     1201 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     1261 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     1321 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     1381 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     1441 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     1501 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     1561 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     1621 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     1681 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     1741 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     1801 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     1861 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     1921 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     1981 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     2041 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
     2101 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     2161 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     2221 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
     2281 acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac
     2341 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
     2401 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     2461 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     2521 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     2581 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     2641 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
     2701 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     2761 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     2821 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     2881 cgcgttttgc ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     2941 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     3001 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     3061 tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga
     3121 ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg
     3181 gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg
     3241 cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag
     3301 gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga
     3361 acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg
     3421 acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt
     3481 agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga
     3541 caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg
     3601 atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat
     3661 aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg
     3721 cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca
     3781 ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct
     3841 ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg
     3901 caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac
     3961 ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact
     4021 gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg
     4081 acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt
     4141 gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg
     4201 gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa attattcata
     4261 atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat
     4321 agagttaggc agggatattc accattatcg tttcagaccc acctcccaac cccgagggga
     4381 cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt
     4441 cgattagtga acggatcggc actgcgtgcg ccaattctgc agacaaatgg cagtattcat
     4501 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     4561 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     4621 ttttcgggtt tattacaggg acagcagaga tccagtttgg ttagtaccgg gcccgctcta
     4681 gccctgcagg aactgtttaa actgtcgtga ccgagcttgt ctgcccgcgc agtccactag
     4741 acagacgcgc agaggtcggg cgggcctagg gccaatttcc catggttccc ttcatttgca
     4801 tatatgatgt aataaggtta tggagactat taaacttagc cctaatcaaa ctatatgatg
     4861 ataatgtgtg tggtacaaaa ggtcataact tattatatac tttgaaactt aaaaaagggt
     4921 tacagtttag tcaccataac tgtaaaattt tttctattct tagctttata tagttcttga
     4981 gaggccatgt ttatggtcat ggctgatgca atgcgggttt aagagctaag ctggaaacag
     5041 catagcaagt ttaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     5101 gctttttttc cagtctgact atcacgattg atccgacgcg ccatctctag gcccgcgccg
     5161 gccccctcgc acggacttgt gggagaagct cggctactcc cctgccccgg ttaatttgca
     5221 tataatattt cctagtaact atagaggctt aatgtgcgat aaaagacaga taatctgttc
     5281 tttttaatac tagctacatt ttacatgata ggcttggatt tctataagag atacaaatac
     5341 taaattatta ttttaaaaaa cagcacaaaa ggaaactcac cctaactgta aagtaattgt
     5401 gtgttttgag actataagta tcccttggag aaccaccttg ttggaagtca agagagccac
     5461 cgaggtttga gagctaagca gaaagctgca tagcaagttc aaataaggct agtccgtaca
     5521 caacttgaaa aagtggcagc cgagtcggct gctttttttg ctcgaatcta cactcagcta
     5581 tggcgcgccc caaggtcggg caggaagagg gcctatttcc catgattcct tcatatttgc
     5641 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     5701 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     5761 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     5821 tggctttata tatcttgtgg aaagccagaa acatgggatc tgctgcacat acggagtttc
     5881 agagctaagc acaagagtgc atagcaagtt gaaataaggc tagtccgttt acaacttgaa
     5941 aaagtggcac ccgagtcggg tgcttttttt gtttggcgcg cctatggctt aattaacatg
     6001 gcggccgcat tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct
     6061 ttagcagccc cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc
     6121 acatccaccg gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct
     6181 actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga
     6241 caaatggaag tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg
     6301 aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct
     6361 cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg
     6421 ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc
     6481 gcgctgttct cctcttcctc atctccggga cccgggatgc ccaagaagaa gaggaaggtg
     6541 tccaatttac tgaccgtaca ccaaaatttg cctgcattac cggtcgatgc aacgagtgat
     6601 gaggttcgca agaacctgat ggacatgttc agggatcgcc aggcgttttc tgagcatacc
     6661 tggaaaatgc ttctgtccgt ttgccggtcg tgggcggcat ggtgcaagtt gaataaccgg
     6721 aaatggtttc ccgcagaacc tgaagatgtt cgcgattatc ttctatatct tcaggcgcgc
     6781 ggtctggcag taaaaactat ccagcaacat ttgggccagc taaacatgct tcatcgtcgg
     6841 tccgggctgc cacgaccaag tgacagcaat gctgtttcac tggttatgcg gcggatccga
     6901 aaagaaaacg ttgatgccgg tgaacgtgca aaacaggctc tagcgttcga acgcactgat
     6961 ttcgaccagg ttcgttcact catggaaaat agcgatcgct gccaggatat acgtaatctg
     7021 gcatttctgg ggattgctta taacaccctg ttacgtatag ccgaaattgc caggatcagg
     7081 gttaaagata tctcacgtac tgacggtggg agaatgttaa tccatattgg cagaacgaaa
     7141 acgctggtta gcaccgcagg tgtagagaag gcacttagcc tgggggtaac taaactggtc
     7201 gagcgatgga tttccgtctc tggtgtagct gatgatccga ataactacct gttttgccgg
     7261 gtcagaaaaa atggtgttgc cgcgccatct gccaccagcc agctatcaac tcgcgccctg
     7321 gaagggattt ttgaagcaac tcatcgattg atttacggcg ctaaggatga ctctggtcag
     7381 agatacctgg cctggtctgg acacagtgcc cgtgtcggag ccgcgcgaga tatggcccgc
     7441 gctggagttt caataccgga gatcatgcaa gctggtggct ggaccaatgt aaatattgtc
     7501 atgaactata tccgtaacct ggatagtgaa acaggggcaa tggtgcgcct gctggaagat
     7561 ggcgattagc catgtttaag ggttccggtt ccactaggta caattcgata tcaagcttat
     7621 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     7681 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     7741 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     7801 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     7861 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     7921 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     7981 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     8041 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     8101 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     8161 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata
     8221 ccgtcgacct cgatcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag
     8281 ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag
     8341 tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact
     8401 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatcc
     8461 ttgatctgtg gatctaccac acacaaggct acttccctga ttggcagaac tacacaccag
     8521 ggccagggat cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc
     8581 aagagaaggt agaagaagcc aatgaaggag agaacacccg cttgttacac cctgtgagcc
     8641 tgcatgggat ggatgacccg gagagagaag tat
//
