LOCUS       sequence-396629-        3518 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains gRNA for CDX4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3518)
  AUTHORS   Maimaitili M, Chen M, Febbraro F, Ucuncu E, Kelly R, Niclis JC, 
            Christiansen JR, Mermet-Joret N, Niculescu D, Lauritsen J, Iannielli
            A, Klaestrup IH, Jensen UB, Qvist P, Nabavi S, Broccoli V, Nykjaer 
            A, Romero-Ramos M, Denham M
  TITLE     Enhanced production of mesencephalic dopaminergic neurons from 
            lineage-restricted human undifferentiated stem cells.
  JOURNAL   Nat Commun. 2023 Dec 5;14(1):7871. doi: 10.1038/s41467-023-43471-0.
  PUBMED    38052784
REFERENCE   2  (bases 1 to 3518)
  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: "Nat 
            Commun."; date: "2023-12-5"; volume: "14(1):7871. doi"; pages: " 
            10.1038/s41467-023-43471-0"
COMMENT     Sequence Label: phU6-CDX4-gRNA
FEATURES             Location/Qualifiers
     source          1..3518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        696..944
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     696..716
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     867..886
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        966..1041
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1097..1117)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1097..1115)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1136..1152)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1136..1152)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1149..1171)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1160..1176
                     /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)."
     promoter        complement(join(1184..1190,1191..1208,1209..1214))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1229..1250
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1367..1384)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1538..2126)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1618..1637)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2203..2997)
                     /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(2203..2997)
                     /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(2314..2333)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2924..2943
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3032..3389)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3046..3181
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        3198..3389
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     3346..3366
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3391..3495)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtacccg
      661 tctcacaggc ggatcgatcc aaggtcgggc aggaagaggg cctatttccc atgattcctt
      721 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
      781 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      841 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      901 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgcctgg gcctttccga
      961 gagacgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     1021 aaagtggcac cgagtcggtg ctttttttct ccgctgagcg tactgagacg ccgcggtgga
     1081 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
     1141 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     1201 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     1261 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     1321 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     1381 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     1441 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     1501 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     1561 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     1621 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     1681 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     1741 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     1801 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     1861 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     1921 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     1981 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2041 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2101 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     2161 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gatcagaaga actcgtcaag
     2221 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
     2281 gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc
     2341 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     2401 ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc
     2461 gggcatgctc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc
     2521 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg
     2581 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     2641 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg
     2701 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     2761 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcttgcag
     2821 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     2881 cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     2941 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg
     3001 aaacgatcct catcctgtct cttgatcgat ctttgcaaaa gcctaggcct ccaaaaaagc
     3061 ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa
     3121 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
     3181 tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac
     3241 ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca
     3301 tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca
     3361 cattccacag ctggttcttt ccgcctcagg actcttcctt tttcaatatt attgaagcat
     3421 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     3481 aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
