LOCUS       sequence-384081-        7080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNA to canine NHERF1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7080)
  AUTHORS   Bryant DM, Roignot J, Datta A, Overeem AW, Kim M, Yu W, Peng X, 
            Eastburn DJ, Ewald AJ, Werb Z, Mostov KE
  TITLE     A molecular switch for the orientation of epithelial cell 
            polarization.
  JOURNAL   Dev Cell. 2014 Oct 27;31(2):171-87. doi: 
            10.1016/j.devcel.2014.08.027. Epub 2014 Oct 9.
  PUBMED    25307480
REFERENCE   2  (bases 1 to 7080)
  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"; doi: 
            "10.1016/j.devcel.2014.08.027"; journalName: "Dev Cell"; date: 
            "2014-10-27- 27"; volume: "31"; issue: "2"; pages: "171-87"
COMMENT     Sequence Label: pLKO-cfNHERF1_4
FEATURES             Location/Qualifiers
     source          1..7080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        142..390
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     142..162
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     313..332
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    483..600
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        649..1159
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1078..1096
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1181..1780
                     /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(1181..1200)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1677..1697
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1908..2141
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2213..2334
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2250..2269)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2304..2323
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2374..2509
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2529..2548)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2530..2548)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2558..2575)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2558..2574)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2567..2589)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2716..3171
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2803..2822)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3013..3034
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3197..3301
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3302..3370,3371..4162)
                     /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(3520..3539)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4333..4921
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4822..4841
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5075..5092
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5209..5230
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5245..5250,5251..5268,5269..5275)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5283..5299
                     /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     5288..5310
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5307..5323
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5307..5323
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5342..5362
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5344..5362
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5390..5616
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5617..5797
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5844..5969
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6462..6695
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6880..6924
                     /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"
ORIGIN
        1 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
       61 agagacagat ccattcgatt agtgaacgga tctcgacggt atcgatcacg agactagcct
      121 cgagcggccg cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata
      181 cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta
      241 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      301 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      361 ttatatatct tgtggaaagg acgaaacacc ggtggggaga tccggaagga gctcgagctc
      421 cttccggatc tccccatttt tgaattctcg acctcgagac aaatggcagt attcatccac
      481 aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
      541 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
      601 cgggtttatt acagggacag cagagatcca ctttggccgc ggctcgaggg ggttggggtt
      661 gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc
      721 cgggaaacgc agcggcgccg accctgggtc tcgcacattc ttcacgtccg ttcgcagcgt
      781 cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg ctccgcccct
      841 aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg
      901 tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc gccgaccgcg
      961 atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg gccgggaagg
     1021 ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg
     1081 ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat
     1141 caccgacctc tctccccagg gggatccacc ggagcttacc atgaccgagt acaagcccac
     1201 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     1261 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     1321 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     1381 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     1441 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     1501 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     1561 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     1621 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     1681 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     1741 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca
     1801 gcgcccgacc gaaaggagcg cacgacccca tgcatcggta cctttaagac caatgactta
     1861 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     1921 tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca
     1981 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     2041 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     2101 atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct
     2161 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt
     2221 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2281 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     2341 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     2401 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     2461 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac
     2521 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     2581 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     2641 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     2701 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2761 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2821 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2881 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2941 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3001 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3061 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3121 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     3181 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3241 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3301 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3361 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3421 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3481 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3541 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     3601 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     3661 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3721 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3781 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3841 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3901 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3961 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4021 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4081 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4141 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4201 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4261 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4321 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4381 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4441 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4501 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4561 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4621 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4681 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4741 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4801 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4861 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4921 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4981 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5041 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5101 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     5161 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     5221 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     5281 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     5341 cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat
     5401 gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga
     5461 gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta
     5521 ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga
     5581 gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc
     5641 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     5701 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     5761 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
     5821 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
     5881 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
     5941 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
     6001 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
     6061 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
     6121 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
     6181 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
     6241 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
     6301 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     6361 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     6421 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     6481 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     6541 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     6601 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     6661 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     6721 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     6781 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     6841 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     6901 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     6961 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     7021 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
//
