LOCUS       sequence-178032-        6372 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Knockdown human Smad3 expression in human mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6372)
  AUTHORS   Sun J, He H, Xiong Y, Lu S, Shen J, Cheng A, Chang WC, Hou MF, 
            Lancaster JM, Kim M, Yang S
  TITLE     Fascin protein is critical for transforming growth factor beta 
            protein-induced invasion and filopodia formation in spindle-shaped 
            tumor cells.
  JOURNAL   J Biol Chem. 2011 Nov 11;286(45):38865-75. doi: 
            10.1074/jbc.M111.270413. Epub 2011 Sep 13.
  PUBMED    21914811
REFERENCE   2  (bases 1 to 6372)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1074/jbc.M111.270413"; journalName: "J Biol Chem"; date: 
            "2011-11-11- 11"; volume: "286"; issue: "45"; pages: "38865-75"
COMMENT     Sequence Label: pSUPER-retro-puro-Smad3
FEATURES             Location/Qualifiers
     source          1..6372
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..215
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     136..155
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(284..300)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(285..301)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     CDS             complement(319..735)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(335..351)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(367..389)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    802..1143
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     LTR             1207..1723
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(1923..1942)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(2070..2092)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2286..2305)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2405..2427
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2465..2483)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2551..2655
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2656..2724,2725..3516)
                     /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(2874..2893)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3687..4275
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4176..4195
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4429..4446
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4563..4584
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4599..4604,4605..4622,4623..4629)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4637..4653
                     /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)."
     CDS             complement(5239..5838)
                     /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(5322..5342)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     5819..5838
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(5863..6362)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5911..5930)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     6304..6327
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
ORIGIN
        1 gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa
       61 cacccagcgc gcgtgcgccc tggcaggaag atggctgtga gggacagggg agtggcgccc
      121 tgcaatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg
      181 gatttgggaa tcttataagt tctgtatgag accacagatc cccggacgag gtctgcgtga
      241 atttcaagag aattcacgca gacctcgtcc tttttaagct tatcgatacc gtcgacctcg
      301 agagatcgat ctaattccgg cgcctagaga aggagtgagg gctggataaa gggaggatcg
      361 aggcggggtc gaacgaggag gttcaagggg gagagacggg gcggatggag gaagaggagg
      421 cggaggctta gggtgtacaa agggcttgac ccagggaggg gggtcaaaag ccaaggcttc
      481 ccaggtcacg atgtagggga cctggtctgg gtgtccatgc gggccaggtg aaaagacctt
      541 gatcttaacc tgggtgatga ggtctcggtt aaaggtgccg tctcgcggcc atccgacgtt
      601 aaaggttggc cattctgcag agcagaaggt aacccaacgt ctcttcttga catctaccga
      661 ctggttgtga gcgatccgct cgacatcttt ccagtgacct aaggtcaaac ttaagggagt
      721 ggtaacagtc tggccctaat tttcagacaa atacagaaac acagtcagac agagacaaca
      781 cagaacgatg ctgcagcgct gcagcagaca agacgcgcgg cttcggttcc aaaccgaaag
      841 caaaaattca gacggaggcg ggaactgttt taggttctcg tctcctacca gaaccacata
      901 tcctgacggg gtcggattcc acatcgactc ccttcctcag gtcgggccac aaaaacggcc
      961 cccaaagtcc ctgggacgtc tcccagggtt gcggccgggt gttcagaact cgtcagttcc
     1021 accacgggtc cgccagatac agagctagtt agctaactag tacagacgca ggcgcaaaca
     1081 ttagatgccg gcacaaacac gcacaaagac agagacagac acgaaacgac cgctggccag
     1141 cttacctccc ggcggggggg tcggtggtcc ctgggcaggg gtctccaaat ctcggtggaa
     1201 cctccaaatg aaagaccccc gaggtgggca gtcaatcaat ctgaggagac cctcccaagg
     1261 atcagcgagt ccacgattcg gatgcaaaca gcaagaggct ttattgggaa tacgggtacc
     1321 cgggcgacgc agtctatcgg aggactggcg cgccgagtga ggggttgtgg gctcttttat
     1381 tgagctcggg gagcagaagc gcgcgaacag aagcgagaag cgaactgatt ggttagttca
     1441 aataaggcac agggtcattt caggtccttg gggcaccctg gaaacatctg atggttctct
     1501 agaaactgct gagggcggga ccgcatctgg ggaccatctg ttcttggccc tgagccgggg
     1561 caggaactgc ttaccacaga tatcctgttt ggcccatatt ctgctgtctc tctgttccta
     1621 accttgatct gaacttctct attctcagtt atgtattttc catgccttgc aaaatggcgt
     1681 tacttaagct agcttgccaa acctacaggt ggggtctttc attcccccct ttttctggag
     1741 actaaataaa atcttttatt ttatctatgg ctcgtactct ataggcttca gctggtgata
     1801 ttgttgagtc aaaactagag cctggaccac tgatatcctg tctttaacaa attggactaa
     1861 tcgcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     1921 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
     1981 cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
     2041 ctccttgcat gcaccattcc ttgcgccgtc gttttacaac gtcgtgactg ggaaaaccct
     2101 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     2161 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     2221 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
     2281 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     2341 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     2401 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     2461 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     2521 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2581 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2641 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2701 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2761 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2821 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2881 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     2941 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3001 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3061 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3121 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3181 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3241 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3301 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3361 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3421 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3481 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3541 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3601 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3661 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3721 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3781 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     3841 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3901 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3961 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4021 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4081 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4141 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4201 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4261 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4321 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4381 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4441 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     4501 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     4561 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     4621 cgtatgttgt gtggaattgt gagcggataa caatttcaca attctcccca gctctttctg
     4681 cgctgtccat aatgaactat ttcaggagta ttggagtatg tggattcaaa acagtggcta
     4741 attgagtcag gattacttaa tattcgactc ctaccctcag aatgttgttc tccaacttca
     4801 agaaactgtt acccatgaaa gacccccgct gacgggtagt caatcactca gaggagaccc
     4861 tcccaaggaa cagcgagacc acaagtcgga tgcaactgca agagggttta ttggatacac
     4921 gggtacccgg gcgacgcagt ctatcggagg actggcgcgc cgagtgaggg gttgtgggct
     4981 cttttattga gctcggggag cagaagcgcg cgaacagaag cgagaagcga actgattggt
     5041 tagttcaaat aaggcacagg gtcatttcag gtccttgggg caccctggaa acatctgatg
     5101 gttctctagc ttgccaaacc tacaggtggg gtctttcatt cccccctttt tctggagact
     5161 aaataaaatc ttttatttta tcgatgcatg gggtcgtgcg ctcctttcgg tcgggcgctg
     5221 cgggtcgtgg ggcgggcgtc aggcaccggg cttgcgggtc atgcaccagg tgcgcggtcc
     5281 ttcgggcacc tcgacgtcgg cggtgacggt gaagccgagc cgctcgtaga aggggaggtt
     5341 gcggggcgcg gaggtctcca ggaaggcggg caccccggcg cgctcggccg cctccactcc
     5401 ggggagcacg acggcgctgc ccagaccctt gccctggtgg tcgggcgaga cgccgacggt
     5461 ggccaggaac cacgcgggct ccttgggccg gtgcggcgcc aggaggcctt ccatctgttg
     5521 ctgcgcggcc agccgggaac cgctcaactc ggccatgcgc gggccgatct cggcgaacac
     5581 cgcccccgct tcgacgctct ccggcgtggt ccagaccgcc accgcggcgc cgtcgtccgc
     5641 gacccacacc ttgccgatgt cgagcccgac gcgcgtgagg aagagttctt gcagctcggt
     5701 gacccgctcg atgtggcggt ccggatcgac ggtgtggcgc gtggcggggt agtcggcgaa
     5761 cgcggcggcg agggtgcgta cggccctggg gacgtcgtcg cgggtggcga ggcgcaccgt
     5821 gggcttgtac tcggtcatgg taagctagct tgggctgcag gtcgaaaggc ccggagatga
     5881 ggaagaggag aacagcgcgg cagacgtgcg cttttgaagc gtgcagaatg ccgggcctcc
     5941 ggaggacctt cgggcgcccg ccccgcccct gagcccgccc ctgagcccgc ccccggaccc
     6001 accccttccc agcctctgag cccagaaagc gaaggagcaa agctgctatt ggccgctgcc
     6061 ccaaaggcct acccgcttcc attgctcagc ggtgctgtcc atctgcacga gactagtgag
     6121 acgtgctact tccatttgtc acgtcctgca cgacgcgagc tgcggggcgg gggggaactt
     6181 cctgactagg ggaggagtag aaggtggcgc gaaggggcca ccaaagaacg gagccggttg
     6241 gcgcctaccg gtggatgtgg aatgtgtgcg aggccagagg ccacttgtgt agcgccaagt
     6301 gcccagcggg gctgctaaag cgcatgctcc agactgcctt gggaaaagcg cctcccctac
     6361 ccggtagaat tc
//
