LOCUS       sequence-363607-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For mammalian expression of shRNA: ATCTTGACTCCCTGAAGTAAC that 
            targets mouse AK1 (adenylate kinase).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7084)
  TITLE     Najafov lab plasmids
REFERENCE   2  (bases 1 to 7084)
  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: pLKO-puro-mAK1-shRNA-1
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     42..59
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    176..197
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(212..217,218..235,236..242)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    250..266
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     255..277
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     274..290
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     274..290
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     309..329
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        311..329
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        357..583
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             584..764
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    811..936
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1429..1662
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1847..1891
                     /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"
     promoter        2189..2437
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2189..2209
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2360..2379
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2534..2651
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2700..3210
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3129..3147
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3232..3831
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3232..3251)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3728..3748
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3959..4192
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4264..4385
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4301..4320)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4355..4374
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4425..4560
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4580..4599)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4581..4599)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4609..4626)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4609..4625)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4618..4640)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4767..5222
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4854..4873)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5064..5085
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5248..5352
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5353..5421,5422..6213)
                     /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     complement(5571..5590)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6384..6972
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6873..6892
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
       61 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
      121 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
      181 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
      241 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
      301 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta
      361 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt
      421 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc
      481 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca
      541 ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga
      601 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
      661 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
      721 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
      781 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
      841 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
      901 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
      961 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
     1021 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
     1081 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     1141 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     1201 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     1261 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     1321 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     1381 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     1441 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     1501 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     1561 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     1621 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     1681 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     1741 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     1801 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     1861 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     1921 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     1981 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     2041 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
     2101 gagagacaga gacagatcca ttcgattagt gaacggatct cgacggtatc gatcacgaga
     2161 ctagcctcga gcggccgccc ccttcaccga gggcctattt cccatgattc cttcatattt
     2221 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
     2281 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     2341 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     2401 cttggcttta tatatcttgt ggaaaggacg aaacaccgga tcttgactcc ctgaagtaac
     2461 ctcgaggtta cttcagggag tcaagatttt ttgaattctc gacctcgaga caaatggcag
     2521 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     2581 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     2641 ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg
     2701 gggttggggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct
     2761 gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc
     2821 gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct
     2881 gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg
     2941 gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg
     3001 cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc
     3061 ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg
     3121 cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc
     3181 gttgaccgaa tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag
     3241 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     3301 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     3361 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     3421 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     3481 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     3541 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     3601 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     3661 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     3721 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     3781 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc
     3841 cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga
     3901 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     3961 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     4021 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     4081 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     4141 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt
     4201 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag
     4261 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4321 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4381 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc
     4441 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4501 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4561 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     4621 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     4681 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     4741 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     4801 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     4861 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4921 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4981 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5041 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     5101 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     5161 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     5221 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5281 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5341 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5401 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5461 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5521 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5581 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5641 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5701 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5761 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5821 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5881 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5941 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6001 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6061 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6121 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6181 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6241 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6301 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6361 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6421 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6481 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6541 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6601 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6661 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6721 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6781 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6841 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6901 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6961 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7021 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     7081 tttg
//
