LOCUS       sequence-148142-        7114 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 7114)
  AUTHORS   Kang Y, He W, Tulley S, Gupta GP, Serganova I, Chen CR, 
            Manova-Todorova K, Blasberg R, Gerald WL, Massague J
  TITLE     Breast cancer bone metastasis mediated by the Smad tumor suppressor 
            pathway.
  JOURNAL   Proc Natl Acad Sci U S A. 2005 Sep 27. 102(39):13909-14.
  PUBMED    16172383
REFERENCE   2  (bases 1 to 7114)
  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: "Proc Natl 
            Acad Sci U S A. 2005 Sep 27. 102(39):13909-14."
COMMENT     Sequence Label: pcDNA Flag-Smad4M
FEATURES             Location/Qualifiers
     source          1..7114
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             744..767
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2471..2488)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2477..2701
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2747..3175
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2834..2853)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3044..3065
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3184..3204)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3189..3518
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3369..3504
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3431..3450
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3585..4379
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3639..3658)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4249..4268
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4553..4674
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4590..4609)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4644..4663
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4723..4739)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4723..4739)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4736..4758)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4747..4763
                     /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(4771..4777,4778..4795,4796..4801))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4816..4837
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4954..4971)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5125..5713)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5205..5224)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5884..6675,6676..6744))
                     /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     6507..6526
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6745..6849)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6924..6943)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacttaagc ttggtaccga gctcggatcc agaattcaga tctggtacca
      721 cgcgtatcga taagcttacc atggactaca aggacgacga tgacaaggac aatatgtcta
      781 ttacgaatac accaacaagt aatgatgcct gtctgagcat tgtgcatagt ttgatgtgcc
      841 atagacaagg tggagagagt gaaacatttg caaaaagagc aattgaaagt ttggtaaaga
      901 agctgaagga gaaaaaagat gaattggatt ctttaataac agctataact acaaatggag
      961 ctcatcctag taaatgtgtt accatacaga gaacattgga tgggaggctt caggtggctg
     1021 gtcggaaagg atttcctcat gtgatctatg cccgtctctg gaggtggcct gatcttcaca
     1081 aaaatgaact aaaacatgtt aaatattgtc agtatgcgtt tgacttaaaa tgtgatagtg
     1141 tctgtgtgaa tccatatcac tacgaacgag ttgtatcacc tggaattgat ctctcaggat
     1201 taacactgca gagtaatgct ccatcaagta tgatggtgaa ggatgaatac gttcatgact
     1261 ttgagggaca gccatcgttg tccactgaag gacattcaat tcaaaccatc cagcatccac
     1321 caagtaatcg tgcatcgaca gagacataca gcaccccagc tctgttagcc ccatctgagt
     1381 ctaatgctac cagcactgcc aactttccca acattcctgt ggcttccaca agtcagcctg
     1441 ccagtatact ggggggcagc catagtgaag gactgttgca gatagcatca gggcctcagc
     1501 caggacagca gcagaatgga tttactggtc agccagctac ttaccatcat aacagcacta
     1561 ccacctggac tggaagtagg actgcaccat acacacctaa tttgcctcac caccaaaacg
     1621 gccatcttca gcaccacccg cctatgccgc cccatcccgg acattactgg cctgttcaca
     1681 atgagcttgc attccagcct cccatttcca atcatcctgc tcctgagtat tggtgttcca
     1741 ttgcttactt tgaaatggat gttcaggtag gagagacatt taaggttcct tcaagctgcc
     1801 ctattgttac tgttgatgga tacgtggacc cttctggagg agatcgcttt tgtttgggtc
     1861 aactctccaa tgtccacagg acagaagcca ttgagagagc aaggttgcac ataggcaaag
     1921 gtgtgcagtt ggaatgtaaa ggtgaaggtg atgtttgggt caggtgcctt agtgaccacg
     1981 cggtctttgt acagagttac tacttagaca gagaagctgg gcgtgcacct ggagatgctg
     2041 ttcataagat ctacccaagt gcatatataa aggtctttga tttgcgtcag tgtcatcgac
     2101 agatgcagca gcaggcggct actgcacaag ctgcagcagc tgcccaggca gcagccgtgg
     2161 caggaaacat ccctggccca ggatcagtag gtggaatagc tccagctatc agtctgtcag
     2221 ctgctgctgg aattggtgtt gatgaccttc gtcgcttatg catactcagg atgagttttg
     2281 tgaaaggctg gggaccggat tacccaagac agagcatcaa agaaacacct tgctggattg
     2341 aaattcactt acaccgggcc ctccagctcc tagacgaagt acttcatacc atgccgattg
     2401 cagacccaca acctttagac tgaggtggat ctagtcgagt ctagagggcc cgtttaaacc
     2461 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     2521 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     2581 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     2641 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     2701 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     2761 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     2821 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2881 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2941 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3001 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3061 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3121 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     3181 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3241 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3301 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3361 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3421 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     3481 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     3541 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     3601 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     3661 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     3721 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     3781 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     3841 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     3901 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     3961 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4021 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4081 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     4141 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4201 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4261 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4321 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4381 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4441 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4501 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     4561 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4621 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4681 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     4741 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4801 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4861 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4921 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4981 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5041 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5101 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5161 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5221 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5281 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5341 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5401 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5461 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5521 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5581 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5641 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5701 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5761 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5821 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5881 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5941 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6001 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6061 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6121 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6181 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6241 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6301 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6361 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6421 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6481 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6541 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6601 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6661 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6721 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6781 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6841 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc
     6901 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     6961 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
     7021 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
     7081 ctgcttcgcg atgtacgggc cagatatacg cgtt
//
