LOCUS       sequence-228463-        6767 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR donor plasmid to tag human transcription factor SMAD3 with 
            GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6767)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 6767)
  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"
COMMENT     Sequence Label: pSMAD3-donor
FEATURES             Location/Qualifiers
     source          1..6767
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(19..36)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(19..35)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(28..50)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(244..263)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     363..385
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(423..441)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        509..613
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(614..682,683..1474)
                     /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(832..851)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1645..2233
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2134..2153
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2387..2404
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2521..2542
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2557..2562,2563..2580,2581..2587)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2595..2611
                     /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     2600..2622
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2619..2635
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2619..2635
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3511..3531
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3544..3588
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             3628..3651
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(3679..3681,3682..3684,3685..4398)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3724..3745)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3985..4004)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(4436..5009,5010..5012,5013..5021)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4602..4619)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4829..4848
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5103..5906
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5166..5185)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5776..5795
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
       61 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
      121 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct
      181 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
      241 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
      301 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
      361 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
      421 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
      481 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
      541 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
      601 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
      661 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
      721 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
      781 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
      841 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
      901 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
      961 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     1021 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     1081 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     1141 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     1201 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     1261 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     1321 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     1381 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     1441 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     1501 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     1561 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     1621 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     1681 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     1741 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     1801 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     1861 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     1921 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     1981 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2041 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2101 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2161 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2221 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     2281 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     2341 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     2401 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     2461 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     2521 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     2581 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     2641 ttacgccaag cttctcagca gatagccttt gctcagtgga ggcctttggg agcctgcctg
     2701 caagctgcgt catcaactag cctgaattta gacaaagtgc tttggggaaa agaaacgtgt
     2761 ggtgtgcctt gtttttttcc agtaattgtt ccatggctcg ttctgcttgt aagattttcc
     2821 aaaataaggc cgtagatgag aattatatca gcacattggg aaccgaaggc atgcggacac
     2881 tgttggcgct ttgtaaaaaa cacccctcaa tcagacagac acacgtcatc gtcgcctgct
     2941 ggggctgggc ccagatttcg agatagggga gcggcacgtg cccgtgtggt acagatggga
     3001 gccaggaagc cctttgctca ggatggaggt ctcctctatt agggggcctg ttacagtaac
     3061 tggtgggcct ctggggtacc ctgcaattga ctttacatat ttaactcttt gccattgact
     3121 ttacatgttt aactctttaa agtcgacttc cttccccctc aagagaactt aatttactca
     3181 aagtcctccc tgggtagagg agtttggccg ggtagtttca gcaagttact ggtaccgctt
     3241 ctaggacagc gtctaacagc atctttggga agatgactgt caccaaagca gaaaaagctt
     3301 tctgacttgt gtaaccccct ggagattttt taagtccccc accccacccc tttccctatt
     3361 tcttacagga gacagactgt gaccagtacc ccctgctgga ttgagctgca cctgaatggg
     3421 cctttgcagt ggcttgacaa ggtcctcacc cagatgggct ccccaagcat ccgctgttcc
     3481 agtgtgtctg ggattccaac tactgcaagc gagaatcttt attttcaggg cgccgccaaa
     3541 ttcaaagaaa ccgctgctgc taaattcgaa cgccagcaca tggacagcgg aggtggaggt
     3601 tcgagcggtc cctcgggttc gtcgagcctg gaagttctgt tccaggggcc cctgtcgtcg
     3661 agcggtccct cgggttcgat ggtgagcaaa ggcgaggagc tgttcaccgg ggtggtgccc
     3721 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     3781 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     3841 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     3901 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     3961 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     4021 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     4081 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     4141 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     4201 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     4261 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     4321 aagcgcgatc acatggtcct taaggagttc gtgaccgccg ccgggatcac tctcggcatg
     4381 gacgagctgt acaagtaatg atctagagtc gagttaatta agaattccgc ccccccccct
     4441 ctccctcccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt
     4501 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc
     4561 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca
     4621 gggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac
     4681 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg
     4741 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt
     4801 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct
     4861 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg
     4921 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt
     4981 ggttttcctt tgaaaaacac gatgataata tggccacaac ctgttacatt gcacaagata
     5041 aaaatatatc atcacgaaca gtaaaactgt ctgcttacat aaacagtaat acaaggggtg
     5101 ttatgggatc ggccattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     5161 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     5221 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     5281 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     5341 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     5401 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     5461 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     5521 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     5581 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     5641 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     5701 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     5761 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     5821 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     5881 atcgccttct tgacgagttc ttctgaagac atcaagtatg gtaggggagg gcaggcttgg
     5941 ggaaaatggc catgcaggag gtggagaaaa ttggaactct actcaaccca ttgttgtcaa
     6001 ggaagaagaa atctttctcc ctcaactgaa ggggtgcacc cacctgtttt ctgaaacaca
     6061 cgagcaaacc cagaggtgga tgttatgaac agctgtgtct gccaaacaca tttacccttt
     6121 ggccccactt tgaagggcaa gaaatggcgt ctgctctggt ggcttaagtg agcagaacag
     6181 gtagtattac accaccggcc ccctcccccc agactctttt tttgagtgac agctttctgg
     6241 gatgtcacag tccaaccaga aacacccctc tgtctaggac tgcagtgtgg agttcacctt
     6301 ggaagggcgt tctaggtagg aagagcccgc agggccatgc agacctcatg cccagctctc
     6361 tgacgcttgt gacagtgcct cttccagtga acattcccag cccagccccg ccccgccccg
     6421 ccccaccact ccagcagacc ttgccccttg tgagctggat agacttggga tggggaggga
     6481 gggagttttg tctgtctccc tcccctctca gaacatactg attgggaggt gcgtgttcag
     6541 cagaacctgc acacaggaca gcgggaaaaa tcgatgagcg ccacctcttt aaaaactcac
     6601 ttacgtttgt cctttttcac tttgaaaagt tggaaggatc tgctgaggcc cagtgcatat
     6661 gcaatgtata gtgtctatta tcacattaat ctcaaagaga ttcgaatgac ggtaagtgtt
     6721 ctcatgaagc aggaggccct tgtcgtggga tggcatttgg tctcagg
//
