LOCUS       sequence-352660-        8672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses neomycin gRNA's ( bU6 and hU6 ), non-targeting gRNA ( mU6 
            ) and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8672)
  TITLE     Winslow lab 9-24-2021 deposit
REFERENCE   2  (bases 1 to 8672)
  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-sgNT-hU6-sgNeo2
FEATURES             Location/Qualifiers
     source          1..8672
                     /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     5606..5626
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5777..5796
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        6016..6515
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6051..6074)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6448..6467
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6519..6539
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6540..7568
                     /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    7624..8212
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7677..7697)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8095..8106)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8215..8231)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8216..8232)
                     /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 ttggcgaggt attcggctcc
     5461 gcggtttgag agctaagcag aaagctgcat agcaagttca aataaggcta gtccgtacac
     5521 aacttgaaaa agtggcagcc gagtcggctg ctttttttgc tcgaatctac actcagctat
     5581 ggcgcgcccc aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca
     5641 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     5701 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     5761 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     5821 ggctttatat atcttgtgga aagccagaaa catggatatt gctgaagagc ttgggtttca
     5881 gagctaagca caagagtgca tagcaagttg aaataaggct agtccgttta caacttgaaa
     5941 aagtggcacc cgagtcgggt gctttttttg tttggcgcgc ctatggctta attaacatgg
     6001 cggccgcatt ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt
     6061 tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca
     6121 catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta
     6181 ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac
     6241 aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga
     6301 agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc
     6361 agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg
     6421 gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg
     6481 cgctgttctc ctcttcctca tctccgggac ccgggatgcc caagaagaag aggaaggtgt
     6541 ccaatttact gaccgtacac caaaatttgc ctgcattacc ggtcgatgca acgagtgatg
     6601 aggttcgcaa gaacctgatg gacatgttca gggatcgcca ggcgttttct gagcatacct
     6661 ggaaaatgct tctgtccgtt tgccggtcgt gggcggcatg gtgcaagttg aataaccgga
     6721 aatggtttcc cgcagaacct gaagatgttc gcgattatct tctatatctt caggcgcgcg
     6781 gtctggcagt aaaaactatc cagcaacatt tgggccagct aaacatgctt catcgtcggt
     6841 ccgggctgcc acgaccaagt gacagcaatg ctgtttcact ggttatgcgg cggatccgaa
     6901 aagaaaacgt tgatgccggt gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt
     6961 tcgaccaggt tcgttcactc atggaaaata gcgatcgctg ccaggatata cgtaatctgg
     7021 catttctggg gattgcttat aacaccctgt tacgtatagc cgaaattgcc aggatcaggg
     7081 ttaaagatat ctcacgtact gacggtggga gaatgttaat ccatattggc agaacgaaaa
     7141 cgctggttag caccgcaggt gtagagaagg cacttagcct gggggtaact aaactggtcg
     7201 agcgatggat ttccgtctct ggtgtagctg atgatccgaa taactacctg ttttgccggg
     7261 tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca gctatcaact cgcgccctgg
     7321 aagggatttt tgaagcaact catcgattga tttacggcgc taaggatgac tctggtcaga
     7381 gatacctggc ctggtctgga cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg
     7441 ctggagtttc aataccggag atcatgcaag ctggtggctg gaccaatgta aatattgtca
     7501 tgaactatat ccgtaacctg gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg
     7561 gcgattagcc atgtttaagg gttccggttc cactaggtac aattcgatat caagcttatc
     7621 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     7681 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     7741 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     7801 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     7861 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     7921 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     7981 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     8041 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     8101 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     8161 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     8221 cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc acaagtagca atacagcagc
     8281 taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt
     8341 cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     8401 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct
     8461 tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact acacaccagg
     8521 gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac cagttgagca
     8581 agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc ctgtgagcct
     8641 gcatgggatg gatgacccgg agagagaagt at
//
