LOCUS       sequence-252065-        7526 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of pR-spondin 1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7526)
  AUTHORS   Urbischek M, Rannikmae H, Foets T, Ravn K, Hyvonen M, de la Roche M
  TITLE     Organoid culture media formulated with growth factors of defined 
            cellular activity.
  JOURNAL   Sci Rep. 2019 Apr 17;9(1):6193. doi: 10.1038/s41598-019-42604-0.
  PUBMED    30996238
REFERENCE   2  (bases 1 to 7526)
  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: "Sci Rep."; 
            date: "2019-04-17"; volume: "9(1):6193. doi"; pages: " 
            10.1038/s41598-019-42604-0"
COMMENT     Sequence Label: pR-spondin 1
FEATURES             Location/Qualifiers
     source          1..7526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..23
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        111..129
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    130..154
                     /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             169..191
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             198..1301
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKL
                     EEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGK
                     LIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLI
                     AADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKG
                     ETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEF
                     LENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSA
                     FWYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1275..1298
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1320..1337
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1344..1388
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1764..1781
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1850..1868
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1869..1893
                     /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             2602..2646
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2684..2702)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2698..2745
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2782..3237
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2869..2888)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3079..3100
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3352..4143,4144..4212))
                     /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     3975..3994
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4213..4305)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4386..4974
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4875..4894
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5128..5145
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5160..5299
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5225..5244)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5385..5407
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5401..5592)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6164..7246)
                     /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     7208..7227
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7247..7324)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
       61 cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat taatacgact
      121 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
      181 taagaaggag atataccatg gcaaaaatcg aagaaggtaa actggtaatc tggattaacg
      241 gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa gataccggaa
      301 ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag gttgcggcaa
      361 ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc tacgctcaat
      421 ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg tatccgttta
      481 cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct gttgaagcgt
      541 tatcgctgat ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc
      601 cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag
      661 aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa
      721 acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga
      781 ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat tactccatcg
      841 cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg tgggcatggt
      901 ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc ttcaagggtc
      961 aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca
     1021 aagagctggc gaaagagttc ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg
     1081 ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga
     1141 aagatccacg tattgccgcc actatggaaa acgcccagaa aggtgaaatc atgccgaaca
     1201 tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg
     1261 gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac taacgcggcg gccgaattcc
     1321 tggttccgcg tggatccggg tccgggttga atgatatttt cgaagcacag aaaattgaat
     1381 ggcatgagag ccgggggatc aaggggaaaa ggcagaggcg gatcagtgcc gaggggagcc
     1441 aggcctgtgc caaaggctgt gagctctgct ctgaagtcaa cggctgcctc aagtgctcac
     1501 ccaagctgtt catcctgctg gagaggaacg acatccgcca ggtgggcgtc tgcttgccgt
     1561 cctgcccacc tggatacttc gacgcccgca accccgacat gaacaagtgc atcaaatgca
     1621 agatcgagca ctgtgaggcc tgcttcagcc ataacttctg caccaagtgt aaggagggct
     1681 tgtacctgca caagggccgc tgctatccag cttgtcccga gggctcctca gctgccaatg
     1741 gcaccatgga gtgcagtagt cctcatcatc accaccatca ttaagcggcc gcataatgct
     1801 taagtcgaac agaaagtaat cgtattgtac acggccgcat aatcgaaatt aatacgactc
     1861 actatagggg aattgtgagc ggataacaat tccccatctt agtatattag ttaagtataa
     1921 gaaggagata tacatatgga tgacgcggca attcaacaaa cgttagccaa aatgggcatc
     1981 aaaagcagcg atattcagcc cgcgcctgta gctggcatga agacagttct gactaacagc
     2041 ggcgtgttgt acatcaccga tgatggtaaa catatcattc aggggccaat gtatgacgtt
     2101 agtggcacgg ctccggtcaa tgtcaccaat aagatgctgt taaagcagtt gaatgcgctt
     2161 gaaaaagaga tgatcgttta taaagcgccg caggaaaaac acgtcatcac cgtgtttact
     2221 gatattacct gtggttactg ccacaaactg catgagcaaa tggcagacta caacgcgctg
     2281 gggatcaccg tgcgttatct tgctttcccg cgccaggggc tggacagcga tgcagagaaa
     2341 gaaatgaaag ctatctggtg tgcgaaagat aaaaacaaag cgtttgatga tgtgatggca
     2401 ggtaaaagcg tcgcaccagc cagttgcgac gtggatattg ccgaccatta cgcacttggc
     2461 gtccagcttg gcgttagcgg tactccggca gttgtgctga gcaatggcac acttgttccg
     2521 ggttaccagc cgccgaaaga gatgaaagag ttcctcgacg aacaccaaaa aatgaccagc
     2581 ggtaaataac tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg
     2641 gactcgtcta ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta
     2701 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     2761 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2821 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2881 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2941 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3001 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3061 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3121 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3181 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg
     3241 gcggcacgat ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3301 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     3361 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3421 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3481 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     3541 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     3601 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     3661 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     3721 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     3781 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     3841 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     3901 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     3961 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4021 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     4081 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     4141 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     4201 ttgaatactc atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc
     4261 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa
     4321 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4381 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4441 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4501 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     4561 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4621 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4681 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4741 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4801 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4861 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4921 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4981 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     5041 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     5101 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     5161 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     5221 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     5281 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5341 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5401 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     5461 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     5521 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     5581 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     5641 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     5701 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     5761 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     5821 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     5881 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     5941 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     6001 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc
     6061 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     6121 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     6181 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6241 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     6301 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     6361 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     6421 tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg
     6481 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     6541 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     6601 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     6661 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     6721 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     6781 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     6841 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     6901 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     6961 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     7021 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     7081 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     7141 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     7201 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     7261 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     7321 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     7381 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     7441 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     7501 ccgaagtggc gagcccgatc ttcccc
//
