LOCUS       sequence-386696-        7080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNA to canine NHERF2..
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-cfNHERF2_5
FEATURES             Location/Qualifiers
     source          1..7080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     409..427
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             512..1111
                     /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(512..531)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1008..1028
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1239..1472
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1544..1665
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1581..1600)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1635..1654
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1705..1840
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1860..1879)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1861..1879)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1889..1906)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1889..1905)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1898..1920)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2047..2502
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2134..2153)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2344..2365
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2528..2632
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2633..2701,2702..3493)
                     /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(2851..2870)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3664..4252
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4153..4172
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4406..4423
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4540..4561
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4576..4581,4582..4599,4600..4606)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4614..4630
                     /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     4619..4641
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4638..4654
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4638..4654
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4673..4693
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4675..4693
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4721..4947
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             4948..5128
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5175..5300
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5793..6026
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6211..6255
                     /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        6553..6801
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6553..6573
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6724..6743
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    6894..7011
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(7060..7080,1..490)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
ORIGIN
        1 ccaaggcagc cctgggtttg cgcagggacg cggctgctct gggcgtggtt ccgggaaacg
       61 cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg tcacccggat
      121 cttcgccgct acccttgtgg gccccccggc gacgcttcct gctccgcccc taagtcggga
      181 aggttccttg cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac gtctcactag
      241 taccctcgca gacggacagc gccagggagc aatggcagcg cgccgaccgc gatgggctgt
      301 ggccaatagc ggctgctcag cagggcgcgc cgagagcagc ggccgggaag gggcggtgcg
      361 ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt gttccgcatt
      421 ctgcaagcct ccggagcgca cgtcggcagt cggctccctc gttgaccgaa tcaccgacct
      481 ctctccccag ggggatccac cggagcttac catgaccgag tacaagccca cggtgcgcct
      541 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
      601 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
      661 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
      721 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
      781 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
      841 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
      901 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
      961 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     1021 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     1081 ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc agcgcccgac
     1141 cgaaaggagc gcacgacccc atgcatcggt acctttaaga ccaatgactt acaaggcagc
     1201 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     1261 acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc
     1321 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
     1381 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
     1441 acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc ttattattca
     1501 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc
     1561 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     1621 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta
     1681 gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     1741 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     1801 gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc
     1861 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     1921 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     1981 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     2041 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2101 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2161 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2221 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2281 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2341 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2401 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     2461 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     2521 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2581 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     2641 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     2701 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     2761 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     2821 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     2881 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     2941 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3001 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     3061 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3121 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     3181 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     3241 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     3301 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     3361 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     3421 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     3481 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     3541 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     3601 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3661 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3721 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3781 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     3841 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3901 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3961 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4021 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4081 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4141 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4201 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4261 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4321 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4381 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     4441 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     4501 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     4561 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     4621 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     4681 ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc
     4741 ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag
     4801 caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa
     4861 cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta
     4921 tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg
     4981 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     5041 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     5101 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag
     5161 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag
     5221 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga
     5281 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa
     5341 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca
     5401 gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac
     5461 aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat
     5521 ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg
     5581 aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg
     5641 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
     5701 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
     5761 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
     5821 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
     5881 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
     5941 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
     6001 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
     6061 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
     6121 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
     6181 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
     6241 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
     6301 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
     6361 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
     6421 aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga
     6481 tccattcgat tagtgaacgg atctcgacgg tatcgatcac gagactagcc tcgagcggcc
     6541 gcccccttca ccgagggcct atttcccatg attccttcat atttgcatat acgatacaag
     6601 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
     6661 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     6721 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     6781 ttgtggaaag gacgaaacac cgggcgcgag atcttcagca acctcgaggt tgctgaagat
     6841 ctcgcgcttt ttgaattctc gacctcgaga caaatggcag tattcatcca caattttaaa
     6901 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca
     6961 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat
     7021 tacagggaca gcagagatcc actttggccg cggctcgagg gggttggggt tgcgcctttt
//
