LOCUS       sequence-234971-        5830 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produce human recombinant FGF2 G3 in DE3 cells. The depositing 
            laboratory recommends Plasmid 135521: pET-28a(+)-FGF2-G3 as an 
            updated version of this plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5830)
  AUTHORS   Kuo HH, Gao X, DeKeyser JM, Fetterman KA, Pinheiro EA, Weddle CJ, 
            Fonoudi H, Orman MV, Romero-Tejeda M, Jouni M, Blancard M, Magdy T, 
            Epting CL, George AL Jr, Burridge PW
  TITLE     Negligible-Cost and Weekend-Free Chemically Defined Human iPSC 
            Culture.
  JOURNAL   Stem Cell Reports. 2020 Jan 9. pii: S2213-6711(19)30446-1. doi: 
            10.1016/j.stemcr.2019.12.007.
  PUBMED    31928950
REFERENCE   2  (bases 1 to 5830)
  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: "Stem Cell 
            Reports. 2020 Jan 9. pii: S2213-6711(19)30446-1. doi: 
            10.1016/j.stemcr.2019.12.007."
COMMENT     Sequence Label: pET-28a(+)-FGF2-G3
FEATURES             Location/Qualifiers
     source          1..5830
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             148..180
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             695..712
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(765..783)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      779..826
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      863..1318
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(950..969)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1160..1181
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1410..2225)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1410..2225)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2133..2152
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2217..2236
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2347..2935
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2836..2855
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3089..3106
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3121..3260
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3186..3205)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3346..3368
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3362..3553)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4362..5444)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5406..5425
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5445..5522)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5728..5747
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gctagcatga ctggtggaca gcaaatgggt
      181 cgcggatcca tggcagcagg ttcgatcact acattaccgg cactgccgga agatggtggt
      241 tcaggtgcat ttcctcctgg ccacttcaaa gaccctaaac tgctgtactg caagaatggc
      301 ggcttctttc tgcgcattca cccggatggc cgtgttgatg gtactcgcga taaatcagat
      361 ccgttcatca agctgcagct gcaagcggaa gaacgtggcg tggtgagcat taagggcgtt
      421 tgtgcaaacc gttatttagc gatgaaggaa gacggccgcc tgtacgcgat caagaacgtg
      481 accgacgaat gcttcttctt tgaacgcctg gaagaaaaca actacaacac ctaccgcagc
      541 cgcaagtacc cgagctggta tgttgcgtta aagcgtaccg gccagtataa attaggcagc
      601 aaaaccggtc cgggccagaa ggcgattctg tttctgccta tgagcgcgaa gtcagaattc
      661 gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc
      721 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
      781 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
      841 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
      901 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
      961 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     1021 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     1081 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     1141 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     1201 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     1261 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     1321 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     1381 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
     1441 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     1501 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     1561 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     1621 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     1681 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     1741 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     1801 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     1861 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     1921 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     1981 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     2041 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     2101 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     2161 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     2221 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     2281 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2341 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2401 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2461 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     2521 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2581 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2641 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     2701 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     2761 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     2821 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     2881 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     2941 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3001 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3061 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3121 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     3181 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     3241 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3301 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3361 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     3421 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     3481 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     3541 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     3601 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     3661 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     3721 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     3781 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     3841 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     3901 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     3961 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     4021 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     4081 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     4141 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     4201 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     4261 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     4321 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     4381 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4441 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     4501 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     4561 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     4621 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     4681 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     4741 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     4801 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     4861 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     4921 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     4981 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     5041 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     5101 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     5161 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     5221 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     5281 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     5341 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     5401 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     5461 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     5521 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     5581 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     5641 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     5701 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     5761 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     5821 cccgcgaaat
//
