LOCUS       sequence-222234-        5922 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For mammalian expression of the human NODAL open reading frame (with
            two mutated N-glycosylation sites) with a C-terminal MYC-DYK tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5922)
  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 5922)
  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: NODAL-MYC-DYK-N72A-N199A
FEATURES             Location/Qualifiers
     source          1..5922
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        118..497
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        498..701
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     652..672
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     727..746
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        727..745
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1863..1892
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1911..1934
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1954..1970)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1954..1970)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1981..2603
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2180..2199)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      complement(2752..3340)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2832..2851)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3669..3716
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3741..3760
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3948..4742)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3948..4742)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4059..4078)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4669..4688
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4777..5134)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4791..4926
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        4943..5134
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5091..5111
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(5136..5240)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5267..5722
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5354..5373)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5564..5585
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5848..5870
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5862..5879
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5863..5879
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tattagtcat tggttatata gcataaatca atattggcta ttggccattg catacgttgt
       61 atctatatca taatatgtac atttatattg gctcatgtcc aatatgaccg ccatgttgac
      121 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      181 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      241 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      301 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      361 tgtatcatat gccaagtccg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      421 attatgccca gtacatgacc ttacgggact ttcctacttg gcagtacatc tacgtattag
      481 tcatcgctat taccatggtg atgcggtttt ggcagtacac caatgggcgt ggatagcggt
      541 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      601 accaaaatca acgggacttt ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg
      661 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
      721 attttgtaat acgactcact atagggcggc cgggaattcg tcgactggat ccggtaccga
      781 ggagatctgc cgccgcgatc gccatgcacg cccactgcct gcccttcctt ctgcacgcct
      841 ggtgggccct actccaggcg ggtgctgcga cggtggccac tgcgctcctg cgtacgcggg
      901 ggcagccctc gtcgccatcc cctctggcgt acatgctgag cctctaccgc gacccgctgc
      961 cgagggcaga catcatccgc agcctacagg cagaagatgt ggcagtggat gggcaggcct
     1021 ggacgtttgc ttttgacttc tccttcctga gccaacaaga ggatctggca tgggctgagc
     1081 tccggctgca gctgtccagc cctgtggacc tccccactga gggctcactt gccattgaga
     1141 ttttccacca gccaaagccc gacacagagc aggcttcaga cagctgctta gagcggtttc
     1201 agatggacct attcactgtc actttgtccc aggtcacctt ttccttgggc agcatggttt
     1261 tggaggtgac caggcctctc tccaagtggc tgaagcgccc tggggccctg gagaagcaga
     1321 tgtccagggt agctggagag tgctggccgc ggccccccac accgcctgcc accaatgtgc
     1381 tccttatgct ctactccgcc ctctcgcagg agcagaggca gctgggtggg tccaccttgc
     1441 tgtgggaagc cgagagctcc tggcgggccc aggagggaca gctgtcctgg gagtggggca
     1501 agaggcaccg tcgacatcac ttgccagaca gaagtcaact gtgtcggaag gtcaagttcc
     1561 aggtggactt caacctgatc ggatggggct cctggatcat ctaccccaag cagtacaacg
     1621 cctatcgctg tgagggcgag tgtcctaatc ctgttgggga ggagtttcat ccgaccaacc
     1681 atgcatacat ccagagtctg ctgaaacgtt accagcccca ccgagtccct tccacttgtt
     1741 gtgccccagt gaagaccaag ccgctgagca tgctgtatgt ggataatggc agagtgctcc
     1801 tagatcacca taaagacatg atcgtggaag aatgtgggtg cctcacgcgt acgcggccgc
     1861 tcgagcagaa actcatctca gaagaggatc tggcagcaaa tgatatcctg gattacaagg
     1921 atgacgacga taaggtttaa acggccggcc gcggtcatag ctgtttcctg aacagatccc
     1981 gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca
     2041 gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc
     2101 ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca
     2161 acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg
     2221 ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg
     2281 ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg
     2341 ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct
     2401 tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg
     2461 attttaaaat aactatacca gcaggaggac gtccagacac agcataggct acctggccat
     2521 gcccaaccgg tgggacattt gagttgcttg cttggcactg tcctctcatg cgttgggtcc
     2581 actcagtaga tgcctgttga attgggtacg cggccagcgg cgagcggtat cagctcactc
     2641 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2701 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2761 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2821 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2881 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2941 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3001 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3061 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3121 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3181 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3241 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3301 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3361 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3421 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3481 aagtatatat gagtaacctg aggctatggc agggcctgcc gccccgacgt tggctgcgag
     3541 ccctgggcct tcacccgaac ttggggggtg gggtggggaa aaggaagaaa cgcgggcgta
     3601 ttggccccaa tggggtctcg gtggggtatc gacagagtgc cagccctggg accgaacccc
     3661 gcgtttatga acaaacgacc caacaccgtg cgttttattc tgtcttttta ttgccgtcat
     3721 agcgcgggtt ccttccggta ttgtctcctt ccgtgtttca gttagcctcc ccctagggtg
     3781 ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa
     3841 aacgattccg aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg
     3901 caggttgggc gtcgcttggt cggtcatttc gaaccccaga gtcccgctca gaagaactcg
     3961 tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg
     4021 aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct
     4081 atgtcctgat agcgatccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg
     4141 ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg
     4201 ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc
     4261 tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg
     4321 atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc
     4381 cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga
     4441 tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg
     4501 agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct
     4561 tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc
     4621 gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag
     4681 ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc
     4741 atgcgaaacg atcctcatcc tgtctcttga tcgatctttg caaaagccta ggcctccaaa
     4801 aaagcctcct cactacttct ggaatagctc agaggccgag gcggcctcgg cctctgcata
     4861 aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg
     4921 cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg
     4981 catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag
     5041 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg
     5101 acacacattc cacagctggt tctttccgcc tcaggactct tcctttttca atattattga
     5161 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5221 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgc gccctgtagc
     5281 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     5341 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     5401 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5461 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     5521 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5581 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     5641 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5701 aaaatattaa cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc
     5761 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     5821 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     5881 ccaagctgat ctatacattg aatcaatatt ggcaattagc ca
//
