LOCUS       sequence-396636-        3547 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains gRNA for CDX1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3547)
  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 3547)
  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: pmU6-CDX1-gRNA
FEATURES             Location/Qualifiers
     source          1..3547
                     /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        670..983
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     902..922
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        1006..1081
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1126..1146)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1126..1144)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1165..1181)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1165..1181)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1178..1200)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1189..1205
                     /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(1213..1219,1220..1237,1238..1243))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1258..1279
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1396..1413)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1567..2155)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1647..1666)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2232..3026)
                     /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(2232..3026)
                     /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(2343..2362)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2953..2972
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3061..3418)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3075..3210
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        3227..3418
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     3375..3395
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3420..3524)
                     /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 tctcagatag atccgacgcc gccatctcta ggcccgcgcc ggccccctcg cacagacttg
      721 tgggagaagc tcggctactc ccctgccccg gttaatttgc atataatatt tcctagtaac
      781 tatagaggct taatgtgcga taaaagacag ataatctgtt ctttttaata ctagctacat
      841 tttacatgat aggcttggat ttctataaga gatacaaata ctaaattatt attttaaaaa
      901 acagcacaaa aggaaactca ccctaactgt aaagtaattg tgtgttttga gactataaat
      961 atcccttgga gaaaagcctt gtttgctaca ccgaccacca acgccgtttt agagctagaa
     1021 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     1081 ctttttttgt actgagacgc cgcggtggag ctccagcttt tgttcccttt agtgagggtt
     1141 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     1201 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     1261 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     1321 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     1381 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     1441 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     1501 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     1561 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     1621 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     1681 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     1741 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     1801 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     1861 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     1921 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     1981 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     2041 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     2101 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     2161 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     2221 tttggtcatg atcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg
     2281 ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca
     2341 gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca
     2401 cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg
     2461 ccatgggtca cgacgagatc ctcgccgtcg ggcatgctcg ccttgagcct ggcgaacagt
     2521 tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct
     2581 tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta
     2641 gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca
     2701 ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc
     2761 cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc
     2821 cacgatagcc gcgctgcctc gtcttgcagt tcattcaggg caccggacag gtcggtcttg
     2881 acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg
     2941 attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct
     3001 gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcgatc
     3061 tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc
     3121 cgaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg
     3181 ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc
     3241 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     3301 acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
     3361 tggggacttt ccacacccta actgacacac attccacagc tggttctttc cgcctcagga
     3421 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3481 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     3541 gtgccac
//
