LOCUS       sequence-352696-        8673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Mark2 (bU6), Mark3 (mU6) and Mark4 (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-sgMark2-mU6-sgMark3-hU6-sgMark4
FEATURES             Location/Qualifiers
     source          1..8673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             153..333
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(395..983)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(475..494)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1154..1945,1946..2014))
                     /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     1777..1796
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2015..2119)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2194..2213)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2385..2764
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2766..2964
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2919..2939
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2982..3162
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    3209..3334
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3831..4064
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4249..4293
                     /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    4591..4708
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     5445..5465
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     5691..5711
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5862..5881
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        6101..6600
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6136..6159)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6533..6552
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6604..6624
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6625..7653
                     /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    7709..8297
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7762..7782)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8180..8191)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8300..8316)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8301..8317)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
ORIGIN
        1 tagaagaagc caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga
       61 tggatgaccc ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc
      121 acatggcccg agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag
      181 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
      241 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
      301 gaccctttta gtcagtgtgg aaaatctcta gcagcatgtg agcaaaaggc cagcaaaagg
      361 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
      421 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
      481 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
      541 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
      601 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
      661 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
      721 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
      781 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
      841 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
      901 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
      961 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     1021 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     1081 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     1141 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     1201 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     1261 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     1321 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     1381 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     1441 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     1501 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     1561 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     1621 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     1681 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     1741 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     1801 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     1861 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     1921 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     1981 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     2041 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     2101 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     2161 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     2221 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     2281 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     2341 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
     2401 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     2461 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     2521 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     2581 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     2641 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     2701 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     2761 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     2821 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     2881 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     2941 acggtgggag gtctatataa gcagcgcgtt ttgcctgtac tgggtctctc tggttagacc
     3001 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     3061 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     3121 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     3181 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     3241 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     3301 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     3361 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     3421 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     3481 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     3541 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     3601 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     3661 cgcacagcaa gcggccggcc gctgatcttc agacctggag gaggagatat gagggacaat
     3721 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     3781 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     3841 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     3901 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     3961 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     4021 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     4081 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     4141 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     4201 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     4261 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     4321 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     4381 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     4441 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     4501 gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg tgcgccaatt
     4561 ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     4621 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     4681 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt
     4741 ttggttagta ccgggcccgc tctagccctg caggaactgt ttaaactgtc gtgaccgagc
     4801 ttgtctgccc gcgcagtcca ctagacagac gcgcagaggt cgggcgggcc tagggccaat
     4861 ttcccatggt tcccttcatt tgcatatatg atgtaataag gttatggaga ctattaaact
     4921 tagccctaat caaactatat gatgataatg tgtgtggtac aaaaggtcat aacttattat
     4981 atactttgaa acttaaaaaa gggttacagt ttagtcacca taactgtaaa attttttcta
     5041 ttcttagctt tatatagttc ttgagaggcc atgtttatgg agtaagtcca acatgctgcg
     5101 gtttaagagc taagctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac
     5161 ttgaaaaagt ggcaccgagt cggtgctttt tttccagtct gactatcacg attgatccga
     5221 cgcgccatct ctaggcccgc gccggccccc tcgcacggac ttgtgggaga agctcggcta
     5281 ctcccctgcc ccggttaatt tgcatataat atttcctagt aactatagag gcttaatgtg
     5341 cgataaaaga cagataatct gttcttttta atactagcta cattttacat gataggcttg
     5401 gatttctata agagatacaa atactaaatt attattttaa aaaacagcac aaaaggaaac
     5461 tcaccctaac tgtaaagtaa ttgtgtgttt tgagactata agtatccctt ggagaaccac
     5521 cttgttggac acatcctcac aggccgaggt ttgagagcta agcagaaagc tgcatagcaa
     5581 gttcaaataa ggctagtccg tacacaactt gaaaaagtgg cagccgagtc ggctgctttt
     5641 tttgctcgaa tctacactca gctatggcgc gccccaaggt cgggcaggaa gagggcctat
     5701 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     5761 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     5821 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     5881 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagcc agaaacatgg
     5941 gactcaacca ccccaacatg tttcagagct aagcacaaga gtgcatagca agttgaaata
     6001 aggctagtcc gtttacaact tgaaaaagtg gcacccgagt cgggtgcttt ttttgtttgg
     6061 cgcgcctatg gcttaattaa catggcggcc gcattctacc gggtagggga ggcgcttttc
     6121 ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt
     6181 ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt
     6241 ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg
     6301 cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag
     6361 atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag
     6421 ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc
     6481 aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc
     6541 acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggacccggg
     6601 atgcccaaga agaagaggaa ggtgtccaat ttactgaccg tacaccaaaa tttgcctgca
     6661 ttaccggtcg atgcaacgag tgatgaggtt cgcaagaacc tgatggacat gttcagggat
     6721 cgccaggcgt tttctgagca tacctggaaa atgcttctgt ccgtttgccg gtcgtgggcg
     6781 gcatggtgca agttgaataa ccggaaatgg tttcccgcag aacctgaaga tgttcgcgat
     6841 tatcttctat atcttcaggc gcgcggtctg gcagtaaaaa ctatccagca acatttgggc
     6901 cagctaaaca tgcttcatcg tcggtccggg ctgccacgac caagtgacag caatgctgtt
     6961 tcactggtta tgcggcggat ccgaaaagaa aacgttgatg ccggtgaacg tgcaaaacag
     7021 gctctagcgt tcgaacgcac tgatttcgac caggttcgtt cactcatgga aaatagcgat
     7081 cgctgccagg atatacgtaa tctggcattt ctggggattg cttataacac cctgttacgt
     7141 atagccgaaa ttgccaggat cagggttaaa gatatctcac gtactgacgg tgggagaatg
     7201 ttaatccata ttggcagaac gaaaacgctg gttagcaccg caggtgtaga gaaggcactt
     7261 agcctggggg taactaaact ggtcgagcga tggatttccg tctctggtgt agctgatgat
     7321 ccgaataact acctgttttg ccgggtcaga aaaaatggtg ttgccgcgcc atctgccacc
     7381 agccagctat caactcgcgc cctggaaggg atttttgaag caactcatcg attgatttac
     7441 ggcgctaagg atgactctgg tcagagatac ctggcctggt ctggacacag tgcccgtgtc
     7501 ggagccgcgc gagatatggc ccgcgctgga gtttcaatac cggagatcat gcaagctggt
     7561 ggctggacca atgtaaatat tgtcatgaac tatatccgta acctggatag tgaaacaggg
     7621 gcaatggtgc gcctgctgga agatggcgat tagccatgtt taagggttcc ggttccacta
     7681 ggtacaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag
     7741 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     7801 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     7861 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     7921 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     7981 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     8041 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     8101 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     8161 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     8221 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     8281 ttgggccgcc tccccgcatc gataccgtcg acctcgatcg agacctagaa aaacatggag
     8341 caatcacaag tagcaataca gcagctacca atgctgattg tgcctggcta gaagcacaag
     8401 aggaggagga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca atgacttaca
     8461 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     8521 actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc
     8581 ctgattggca gaactacaca ccagggccag ggatcagata tccactgacc tttggatggt
     8641 gctacaagct agtaccagtt gagcaagaga agg
//
