LOCUS       sequence-362793-        6376 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6376)
  AUTHORS   Gao S, Alarcon C, Sapkota G, Rahman S, Chen PY, Goerner N, Macias 
            MJ, Erdjument-Bromage H, Tempst P, Massague J
  TITLE     Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-beta 
            signaling.
  JOURNAL   Mol Cell. 2009 Nov 13. 36(3):457-68.
  PUBMED    19917253
REFERENCE   2  (bases 1 to 6376)
  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: "Mol Cell. 
            2009 Nov 13. 36(3):457-68."
COMMENT     Sequence Label: pRS-puro shNEDD4L #1
FEATURES             Location/Qualifiers
     source          1..6376
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..517
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    581..922
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             989..1405
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1335..1357
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1373..1389
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     1423..1439
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1424..1440
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(1513..1727)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(1573..1592)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     promoter        1738..2237
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1773..1796)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2170..2189
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2262..2861
                     /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(2262..2281)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2758..2778
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    3447..3463
                     /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)."
     promoter        complement(join(3471..3477,3478..3495,3496..3501))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3516..3537
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3654..3671)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3825..4413)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3905..3924)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4584..5375,5376..5444))
                     /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     5207..5226
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5445..5549)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5617..5635
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5673..5695)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5795..5814
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6008..6030
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6158..6177
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
      121 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
      181 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt
      241 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
      301 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
      361 ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtattcccaa
      421 taaagcctct tgctgtttgc atccgaatcg tggactcgct gatccttggg agggtctcct
      481 cagattgatt gactgcccac ctcgggggtc tttcatttgg aggttccacc gagatttgga
      541 gacccctgcc cagggaccac cgaccccccc gccgggaggt aagctggcca gcggtcgttt
      601 cgtgtctgtc tctgtctttg tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct
      661 gtactagtta gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg
      721 aacacccggc cgcaaccctg ggagacgtcc cagggacttt gggggccgtt tttgtggccc
      781 gacctgagga agggagtcga tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg
      841 agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg
      901 aagccgcgcg tcttgtctgc tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac
      961 tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac
     1021 cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa
     1081 gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg
     1141 agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg
     1201 cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga
     1261 cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc
     1321 cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct ccctttatcc
     1381 agccctcact ccttctctag gcgccggaat tagatcgatc tctcgaggtc gacggtatcg
     1441 ataagctttt ccaaaaagct agactgtgga ttgagttctc ttgaaactca atccacagtc
     1501 tagcggggat ctgtggtctc atacagaact tataagattc ccaaatccaa agacatttca
     1561 cgtttatggt gatttcccag aacacatagc gacatgcaaa tattgcaggg cgccactccc
     1621 ctgtccctca cagccatctt cctgccaggg cgcacgcgcg ctgggtgttc ccgcctagtg
     1681 acactgggcc cgcgattcct tggagcgggt tgatgacgtc agcgttcgaa ttctaccggg
     1741 taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca
     1801 cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc
     1861 aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga
     1921 agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc
     1981 tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg
     2041 gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg
     2101 ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg
     2161 gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct
     2221 catctccggg cctttcgacc tgcagcccaa gctagcttac catgaccgag tacaagccca
     2281 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     2341 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     2401 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     2461 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     2521 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     2581 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     2641 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     2701 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     2761 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     2821 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc
     2881 agcgcccgac cgaaaggagc gcacgacccc atgcatcgat aaaataaaag attttattta
     2941 gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagagaacc
     3001 atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa
     3061 ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga
     3121 gcccacaacc cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg
     3181 tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt tccttgggag
     3241 ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcatgggta acagtttctt
     3301 gaagttggag aacaacattc tgagggtagg agtcgaatat taagtaatcc tgactcaatt
     3361 agccactgtt ttgaatccac atactccaat actcctgaaa tagttcatta tggacagcgc
     3421 agaaagagct ggggagaatt gtgaaattgt tatccgctca caattccaca caacatacga
     3481 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     3541 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3601 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3661 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3721 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3781 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3841 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3901 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3961 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4021 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4081 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4141 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4201 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4261 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4321 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4381 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4441 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4501 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4561 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4621 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4681 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4741 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4801 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4861 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4921 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4981 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5041 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5101 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5161 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5221 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5281 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5341 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5401 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5461 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5521 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     5581 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     5641 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     5701 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     5761 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     5821 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     5881 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     5941 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
     6001 gggttttccc agtcacgacg ttgtaaaacg acggcgcaag gaatggtgca tgcaaggaga
     6061 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     6121 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     6181 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggcgat
     6241 tagtccaatt tgttaaagac aggatatcag tggtccaggc tctagttttg actcaacaat
     6301 atcaccagct gaagcctata gagtacgagc catagataaa ataaaagatt ttatttagtc
     6361 tccagaaaaa gggggg
//
