LOCUS       sequence-178052-        6902 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A lentiviral construct for building stable cell lines expressing GL2
            shRNA via puromycin selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6902)
  AUTHORS   Madugula V, Lu L
  TITLE     A ternary complex comprising transportin1, Rab8 and the ciliary 
            targeting signal directs proteins to ciliary membranes.
  JOURNAL   J Cell Sci. 2016 Oct 15;129(20):3922-3934. Epub 2016 Sep 15.
  PUBMED    27633000
REFERENCE   2  (bases 1 to 6902)
  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: "J Cell 
            Sci."; date: "2016-10-15"; volume: "129(20)"; pages: "3922-3934. 
            Epub 2016 Sep 15"
COMMENT     Sequence Label: GL2 shRNA
FEATURES             Location/Qualifiers
     source          1..6902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..600
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     501..520
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(742..747,748..765,766..772)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    780..796
                     /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     785..807
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     804..820
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     804..820
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     839..859
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        841..859
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        887..1113
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1114..1294
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1341..1466
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1959..2192
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2377..2421
                     /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        2719..2967
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2719..2739
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2890..2909
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    3064..3181
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3230..3740
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3659..3677
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3762..4361
                     /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(3762..3781)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4258..4278
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4489..4722
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4794..4915
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4831..4850)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4885..4904
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4955..5090
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5110..5129)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5111..5129)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5139..5156)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5139..5155)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5148..5170)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5297..5752
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5384..5403)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5594..5615
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5778..5882
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5883..5951,5952..6743)
                     /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(6101..6120)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
       61 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
      121 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
      181 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
      241 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
      301 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
      361 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
      421 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
      481 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
      541 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
      601 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
      661 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      721 aactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      781 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc
      841 aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta gtcttatgca
      901 atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag
      961 aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga
     1021 aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca ttgcagagat
     1081 attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga ccagatctga
     1141 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     1201 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     1261 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
     1321 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
     1381 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
     1441 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
     1501 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
     1561 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
     1621 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1681 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1741 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1801 aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa
     1861 ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag
     1921 agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc
     1981 ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga
     2041 caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa
     2101 cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct
     2161 gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc
     2221 atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt
     2281 tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata
     2341 cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa
     2401 ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata
     2461 aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt
     2521 tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca
     2581 accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga
     2641 gacagatcca ttcgattagt gaacggatct cgacggtatc gatcacgaga ctagcctcga
     2701 gcggccgccc ccttcaccga gggcctattt cccatgattc cttcatattt gcatatacga
     2761 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
     2821 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     2881 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     2941 tatatcttgt ggaaaggacg aaacaccgga acgtacgcgg aatacttcga ctcgagtcga
     3001 agtattccgc gtacgttttt ttgaattctc gacctcgaga caaatggcag tattcatcca
     3061 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     3121 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     3181 tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg gggttggggt
     3241 tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct gggcgtggtt
     3301 ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg
     3361 tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct gctccgcccc
     3421 taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac
     3481 gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg cgccgaccgc
     3541 gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc ggccgggaag
     3601 gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt
     3661 gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc gttgaccgaa
     3721 tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag tacaagccca
     3781 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     3841 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     3901 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     3961 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     4021 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     4081 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     4141 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     4201 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     4261 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     4321 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc
     4381 agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga ccaatgactt
     4441 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     4501 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     4561 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     4621 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     4681 gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc
     4741 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt
     4801 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4861 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4921 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4981 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     5041 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta
     5101 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     5161 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     5221 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     5281 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     5341 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     5401 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5461 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     5521 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5581 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     5641 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5701 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     5761 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5821 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5881 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5941 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     6001 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     6061 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     6121 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     6181 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     6241 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     6301 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6361 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6421 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6481 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6541 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6601 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6661 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6721 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6781 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6841 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6901 tc
//
