LOCUS       sequence-352700-        7769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Same as pLL3.3;U6(BstXI)-XhoI-chimeric RNA;PGK-Cre but XhoI stuffer 
            removed, expresses non-targeting gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7769)
  AUTHORS   Murray CW, Brady JJ, Han M, Cai H, Tsai MK, Pierce SE, Cheng R, 
            Demeter J, Feldser DM, Jackson PK, Shackelford DB, Winslow MM
  TITLE     LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar 
            type II fate in lung cancer.
  JOURNAL   Nat Commun. 2022 Feb 28;13(1):1090. doi: 10.1038/s41467-022-28619-8.
  PUBMED    35228570
REFERENCE   2  (bases 1 to 7769)
  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: "Nat 
            Commun."; date: "2022-02-28"; volume: "13(1):1090. doi"; pages: " 
            10.1038/s41467-022-28619-8"
COMMENT     Sequence Label: hU6-sgNT2 (opti)
FEATURES             Location/Qualifiers
     source          1..7769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(409..420)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(529..545)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(530..546)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             1055..1235
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(1297..1885)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1377..1396)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2056..2847,2848..2916))
                     /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     2679..2698
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2917..3021)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3096..3115)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3287..3666
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3668..3866
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3821..3841
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             3884..4064
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4111..4236
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4733..4966
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5151..5195
                     /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    5493..5610
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5692..5940
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5692..5712
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5863..5882
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        6099..6598
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6134..6157)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6531..6550
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6602..6622
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6623..7651
                     /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    join(7707..7769,1..526)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(join(7760..7769,1..11))
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
ORIGIN
        1 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
       61 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
      121 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
      181 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
      241 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
      301 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
      361 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
      421 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
      481 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga
      541 cctcgatcga gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa
      601 tgctgattgt gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc
      661 tcaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
      721 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct
      781 gtggatctac cacacacaag gctacttccc tgattggcag aactacacac cagggccagg
      841 gatcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg agcaagagaa
      901 ggtagaagaa gccaatgaag gagagaacac ccgcttgtta caccctgtga gcctgcatgg
      961 gatggatgac ccggagagag aagtattaga gtggaggttt gacagccgcc tagcatttca
     1021 tcacatggcc cgagagctgc atccggactg tactgggtct ctctggttag accagatctg
     1081 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     1141 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     1201 cagacccttt tagtcagtgt ggaaaatctc tagcagcatg tgagcaaaag gccagcaaaa
     1261 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     1321 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1381 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1441 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     1501 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     1561 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     1621 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     1681 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     1741 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     1801 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     1861 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     1921 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     1981 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2041 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     2101 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     2161 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     2221 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     2281 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     2341 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     2401 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     2461 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     2521 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     2581 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     2641 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     2701 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     2761 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     2821 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     2881 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     2941 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3001 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     3061 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3121 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     3181 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     3241 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
     3301 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     3361 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     3421 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     3481 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     3541 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     3601 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     3661 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     3721 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     3781 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     3841 gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc tctggttaga
     3901 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     3961 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     4021 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
     4081 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
     4141 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
     4201 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
     4261 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
     4321 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
     4381 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     4441 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     4501 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     4561 accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca
     4621 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     4681 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     4741 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     4801 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     4861 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     4921 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     4981 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     5041 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     5101 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     5161 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     5221 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     5281 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     5341 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     5401 gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg cgtgcgccaa
     5461 ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
     5521 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     5581 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     5641 gtttggttag taccgggccc gctctagccc tgcaggaact gtttaaactg tgagggccta
     5701 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga
     5761 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
     5821 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
     5881 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg accaaacacc
     5941 gacgtagcct tccgaaatat gtttaagagc tatgctggaa acagcatagc aagtttaaat
     6001 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgtttggcg
     6061 cgcctatggc ttaattaaca tggcggccgc attctaccgg gtaggggagg cgcttttccc
     6121 aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg
     6181 cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg
     6241 tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca
     6301 gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat
     6361 ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct
     6421 ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag
     6481 gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac
     6541 gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gacccgggat
     6601 gcccaagaag aagaggaagg tgtccaattt actgaccgta caccaaaatt tgcctgcatt
     6661 accggtcgat gcaacgagtg atgaggttcg caagaacctg atggacatgt tcagggatcg
     6721 ccaggcgttt tctgagcata cctggaaaat gcttctgtcc gtttgccggt cgtgggcggc
     6781 atggtgcaag ttgaataacc ggaaatggtt tcccgcagaa cctgaagatg ttcgcgatta
     6841 tcttctatat cttcaggcgc gcggtctggc agtaaaaact atccagcaac atttgggcca
     6901 gctaaacatg cttcatcgtc ggtccgggct gccacgacca agtgacagca atgctgtttc
     6961 actggttatg cggcggatcc gaaaagaaaa cgttgatgcc ggtgaacgtg caaaacaggc
     7021 tctagcgttc gaacgcactg atttcgacca ggttcgttca ctcatggaaa atagcgatcg
     7081 ctgccaggat atacgtaatc tggcatttct ggggattgct tataacaccc tgttacgtat
     7141 agccgaaatt gccaggatca gggttaaaga tatctcacgt actgacggtg ggagaatgtt
     7201 aatccatatt ggcagaacga aaacgctggt tagcaccgca ggtgtagaga aggcacttag
     7261 cctgggggta actaaactgg tcgagcgatg gatttccgtc tctggtgtag ctgatgatcc
     7321 gaataactac ctgttttgcc gggtcagaaa aaatggtgtt gccgcgccat ctgccaccag
     7381 ccagctatca actcgcgccc tggaagggat ttttgaagca actcatcgat tgatttacgg
     7441 cgctaaggat gactctggtc agagatacct ggcctggtct ggacacagtg cccgtgtcgg
     7501 agccgcgcga gatatggccc gcgctggagt ttcaataccg gagatcatgc aagctggtgg
     7561 ctggaccaat gtaaatattg tcatgaacta tatccgtaac ctggatagtg aaacaggggc
     7621 aatggtgcgc ctgctggaag atggcgatta gccatgttta agggttccgg ttccactagg
     7681 tacaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
     7741 tgactggtat tcttaactat gttgctcct
//
