LOCUS       sequence-274493-        8829 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pLB is a modification of pLL3.7 (Addgene#11795). Genetic elements 
            known to prevent epigenetic silencing were added..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8829)
  AUTHORS   Kissler S, Stern P, Takahashi K, Hunter K, Peterson LB, Wicker LS
  TITLE     In vivo RNA interference demonstrates a role for Nramp1 in modifying
            susceptibility to type 1 diabetes.
  JOURNAL   Nat Genet. 2006 Apr . 38(4):479-83.
  PUBMED    16550170
REFERENCE   2  (bases 1 to 8829)
  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 Genet. 
            2006 Apr . 38(4):479-83."
COMMENT     Sequence Label: pLB
FEATURES             Location/Qualifiers
     source          1..8829
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /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    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3002..3314
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     3233..3253
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     3331..3347
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3389..3422)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        3538..3841
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3842..4045
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      4084..4093
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4090..4092,4093..4095,4096..4809)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4090..4092,4093..4095,4096..4806)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4135..4156)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4396..4415)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4743..4764
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4829..4862)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4918..5506
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4971..4991)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5389..5400)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5509..5525)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             6835..7015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7077..7665)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7157..7176)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7836..8627,8628..8696))
                     /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     8459..8478
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8697..8801)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta gtaccgggcc cggtgctttg ctctgagcca gcccaccagt
     2641 ttggaatgac tcctttttat gacttgaatt ttcaagtata aagtctagtg ctaaatttaa
     2701 tttgaacaac tgtatagttt ttgctggttg ggggaaggaa aaaaaatggt ggcagtgttt
     2761 ttttcagaat tagaagtgaa atgaaaactt gttgtgtgtg aggatttcta atgacatgtg
     2821 gtggttgcat actgagtgaa gccggtgagc attctgccat gtcaccccct cgtgctcagt
     2881 aatgtacttt acagaaatcc taaactcaaa agattgatat aaaccatgct tcttgtgtat
     2941 atccggtctc ttctctgggt agtctcactc agcctgcatt tctgccaggg cccgctctag
     3001 agatccgacg cgccatctct aggcccgcgc cggccccctc gcacagactt gtgggagaag
     3061 ctcggctact cccctgcccc ggttaatttg catataatat ttcctagtaa ctatagaggc
     3121 ttaatgtgcg ataaaagaca gataatctgt tctttttaat actagctaca ttttacatga
     3181 taggcttgga tttctataag agatacaaat actaaattat tattttaaaa aacagcacaa
     3241 aaggaaactc accctaactg taaagtaatt gtgtgttttg agactataaa tatcccttgg
     3301 agaaaagcct tgttaacgcg cggtgaccct cgaggtcgac ggtatcgata agctcgcttc
     3361 acgagattcc agcaggtcga gggacctaat aacttcgtat agcatacatt atacgaagtt
     3421 atattaaggg ttccaagctt aagcggccgc gtggataacc gtattaccgc catgcattag
     3481 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     3541 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     3601 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     3661 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     3721 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     3781 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     3841 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     3901 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     3961 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     4021 gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct agcgctaccg
     4081 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     4141 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     4201 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     4261 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     4321 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     4381 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     4441 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     4501 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     4561 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     4621 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     4681 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     4741 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     4801 tacaagtagg aattcgtcga gggacctaat aacttcgtat agcatacatt atacgaagtt
     4861 atacatgttt aagggttccg gttccactag gtacaattcg atatcaagct tatcgataat
     4921 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     4981 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     5041 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     5101 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     5161 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     5221 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     5281 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     5341 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     5401 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     5461 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga
     5521 cctcgatcga gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa
     5581 tgctgattgt gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc
     5641 tcaggtacca agcatggggt aaagtactgt tctcatcaca tcatatcaag gttatatacc
     5701 atcaatattg ccacagatgt tacttagcct tttaatattt ctctaattta gtgtatatgc
     5761 aatgatagtt ctctgatttc tgagattgag tttctcatgt gtaatgatta tttagagttt
     5821 ctctttcatc tgttcaaatt tttgtctagt tttatttttt actgatttgt aagacttctt
     5881 tttataatct gcatattaca attctcttta ctggggtgtt gcaaatattt tctgtcattc
     5941 tatggcctga cttttcttaa tggtttttta attttaaaaa taagtcttaa tattcatgca
     6001 atctaattaa caatcttttc tttgtggtta ggactttgag tcataagaaa tttttctcta
     6061 cactgaagtc atgatggcat gcttctatat tattttctaa aagatttaaa gttttgcctt
     6121 ctccatttag acttataatt cactggaatt tttttgtgtg tatggtatga catatgggtt
     6181 cccttttatt ttttacatat aaatatattt ccctgttttt ctaaaaaaga aaaagatcat
     6241 cattttccca ttgtaaaatg ccatattttt ttcataggtc acttacatat atcaatgggt
     6301 ctgtttctga gctctactct attttatcag cctcactgtc tatccccaca catctcatgc
     6361 tttgctctaa atcttgatat ttagtggaac attctttccc attttgttct acaagaatat
     6421 ttttgttatt gtcttttggg cttctatata cattttagaa tgaggttggc aagttctgta
     6481 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     6541 agacaagata tccttgatct gtggatctac cacacacaag gctacttccc tgattggcag
     6601 aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg ctacaagcta
     6661 gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac ccgcttgtta
     6721 caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga gtggaggttt
     6781 gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg tactgggtct
     6841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     6901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     6961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagcatg
     7021 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     7081 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     7141 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     7201 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     7261 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     7321 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     7381 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     7441 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     7501 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     7561 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     7621 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     7681 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     7741 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     7801 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     7861 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     7921 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     7981 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     8041 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     8101 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     8161 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     8221 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     8281 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     8341 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     8401 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     8461 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     8521 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     8581 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     8641 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     8701 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     8761 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     8821 ccacctgac
//
