LOCUS       sequence-222467-        5613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For bacterial recombinant protein production of the mature peptide 
            of a human NODAL splice variant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5613)
  AUTHORS   Findlay SD, Bilyk O, Lypka K, Waskiewicz AJ, Postovit L
  TITLE     Genetically regulated human NODAL splice variants are differentially
            post-transcriptionally processed and functionally distinct
  JOURNAL   (2018) bioRxiv 276170
REFERENCE   2  (bases 1 to 5613)
  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: "(2018) 
            bioRxiv 276170"
COMMENT     Sequence Label: pET28a-matNODALvar-HIS
FEATURES             Location/Qualifiers
     source          1..5613
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..43
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     127..146
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        127..145
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    146..170
                     /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             185..207
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             226..243
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             253..270
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             604..621
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(674..692)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      688..735
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      772..1227
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(859..878)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1069..1090
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1319..2134)
                     /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(1319..2134)
                     /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     2042..2061
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2126..2145
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2256..2844
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2745..2764
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2998..3015
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3030..3169
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3095..3114)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3255..3277
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3271..3462)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4271..5353)
                     /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     5315..5334
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5354..5431)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
       61 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
      121 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
      181 taattttgtt taactttaag aaggagatat accatgggca gcagccatca tcatcatcat
      241 cacagcagcg gcctggtgcc gcgcggcagc catatggcta gccatcactt gccagacaga
      301 agtcaactgt gtcggaaggt caagttccag gtggacttca acctgatcgg atggggctcc
      361 tggatcatct accccaagca gtacaacgcc tatcgctgtg agggcgagtg tcctaatcct
      421 gttggggagg agtttcatcc gaccaaccat gcatacatcc aggtggccct gccctgctgt
      481 ccaaggtcat atgggaccaa aatgttttca ttttactcca tgaagtctgg aatgagaatt
      541 tcttggactt gcaatatatc ctccatgcca agcctcagag tctgctgagc ggccgcactc
      601 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
      661 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      721 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
      781 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      841 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
      901 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
      961 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1021 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1081 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1141 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1201 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     1261 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     1321 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     1381 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     1441 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     1501 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     1561 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     1621 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     1681 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     1741 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     1801 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     1861 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     1921 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     1981 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     2041 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     2101 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     2161 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     2221 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2281 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2341 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2401 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2461 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2521 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2581 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2641 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2701 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2761 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2821 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2881 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     2941 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3001 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     3061 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3121 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     3181 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     3241 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     3301 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     3361 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     3421 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     3481 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     3541 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     3601 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     3661 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     3721 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     3781 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     3841 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     3901 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     3961 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     4021 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     4081 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     4141 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     4201 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     4261 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4321 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4381 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4441 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4501 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     4561 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4621 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     4681 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4741 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4801 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4861 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     4921 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     4981 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     5041 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     5101 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     5161 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     5221 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     5281 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5341 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     5401 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     5461 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     5521 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     5581 cccacgccga aacaagcgct catgagcccg aag
//
