LOCUS       sequence-352703-        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 neomycin 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-sgNeo2 (opti)
FEATURES             Location/Qualifiers
     source          1..7769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(325..336)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(445..461)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(446..462)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             971..1151
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(1213..1801)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1293..1312)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1972..2763,2764..2832))
                     /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     2595..2614
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2833..2937)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3012..3031)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3203..3582
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3584..3782
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3737..3757
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             3800..3980
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4027..4152
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4649..4882
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5067..5111
                     /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    5409..5526
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5608..5856
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5608..5628
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5779..5798
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        6015..6514
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6050..6073)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6447..6466
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6518..6538
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6539..7567
                     /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(7623..7769,1..442)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7676..7696)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
ORIGIN
        1 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
       61 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
      121 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
      181 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
      241 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
      301 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
      361 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
      421 tccctttggg ccgcctcccc gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca
      481 tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc
      541 acaagaggag gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac
      601 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
      661 aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta
      721 cttccctgat tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg
      781 atggtgctac aagctagtac cagttgagca agagaaggta gaagaagcca atgaaggaga
      841 gaacacccgc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt
      901 attagagtgg aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc
      961 ggactgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     1021 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     1081 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     1141 aatctctagc agcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     1201 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     1261 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     1321 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     1381 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     1441 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     1501 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     1561 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     1621 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     1681 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     1741 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     1801 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     1861 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     1921 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     1981 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     2041 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     2101 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     2161 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     2221 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     2281 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     2341 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     2401 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     2461 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     2521 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     2581 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     2641 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     2701 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     2761 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     2821 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     2881 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     2941 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     3001 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     3061 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
     3121 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
     3181 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     3241 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     3301 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     3361 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     3421 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     3481 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     3541 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     3601 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     3661 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     3721 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     3781 agcgcgtttt gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     3841 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     3901 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     3961 agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca
     4021 gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg
     4081 cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg
     4141 tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta
     4201 aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta
     4261 gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg
     4321 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
     4381 gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta
     4441 gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccggccgc
     4501 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
     4561 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
     4621 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
     4681 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
     4741 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
     4801 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
     4861 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
     4921 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
     4981 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
     5041 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat
     5101 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
     5161 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga
     5221 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
     5281 gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca
     5341 ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc
     5401 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     5461 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     5521 aattttcggg tttattacag ggacagcaga gatccagttt ggttagtacc gggcccgctc
     5581 tagccctgca ggaactgttt aaactgtgag ggcctatttc ccatgattcc ttcatatttg
     5641 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
     5701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     5761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     5821 ttggctttat atatcttgtg gaaaggacca aacaccgata ttgctgaaga gcttgggttt
     5881 aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga
     5941 aaaagtggca ccgagtcggt gcttttttgt ttggcgcgcc tatggcttaa ttaacatggc
     6001 ggccgcattc taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt
     6061 agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac
     6121 atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac
     6181 tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca
     6241 aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa
     6301 gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca
     6361 gaggctggga aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg
     6421 cgcccgaagg tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc
     6481 gctgttctcc tcttcctcat ctccgggacc cgggatgccc aagaagaaga ggaaggtgtc
     6541 caatttactg accgtacacc aaaatttgcc tgcattaccg gtcgatgcaa cgagtgatga
     6601 ggttcgcaag aacctgatgg acatgttcag ggatcgccag gcgttttctg agcatacctg
     6661 gaaaatgctt ctgtccgttt gccggtcgtg ggcggcatgg tgcaagttga ataaccggaa
     6721 atggtttccc gcagaacctg aagatgttcg cgattatctt ctatatcttc aggcgcgcgg
     6781 tctggcagta aaaactatcc agcaacattt gggccagcta aacatgcttc atcgtcggtc
     6841 cgggctgcca cgaccaagtg acagcaatgc tgtttcactg gttatgcggc ggatccgaaa
     6901 agaaaacgtt gatgccggtg aacgtgcaaa acaggctcta gcgttcgaac gcactgattt
     6961 cgaccaggtt cgttcactca tggaaaatag cgatcgctgc caggatatac gtaatctggc
     7021 atttctgggg attgcttata acaccctgtt acgtatagcc gaaattgcca ggatcagggt
     7081 taaagatatc tcacgtactg acggtgggag aatgttaatc catattggca gaacgaaaac
     7141 gctggttagc accgcaggtg tagagaaggc acttagcctg ggggtaacta aactggtcga
     7201 gcgatggatt tccgtctctg gtgtagctga tgatccgaat aactacctgt tttgccgggt
     7261 cagaaaaaat ggtgttgccg cgccatctgc caccagccag ctatcaactc gcgccctgga
     7321 agggattttt gaagcaactc atcgattgat ttacggcgct aaggatgact ctggtcagag
     7381 atacctggcc tggtctggac acagtgcccg tgtcggagcc gcgcgagata tggcccgcgc
     7441 tggagtttca ataccggaga tcatgcaagc tggtggctgg accaatgtaa atattgtcat
     7501 gaactatatc cgtaacctgg atagtgaaac aggggcaatg gtgcgcctgc tggaagatgg
     7561 cgattagcca tgtttaaggg ttccggttcc actaggtaca attcgatatc aagcttatcg
     7621 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     7681 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     7741 gtatggcttt cattttctcc tccttgtat
//
