LOCUS       sequence-399466-        3567 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains gRNA for CDX2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3567)
  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 3567)
  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: ph7SK-CDX2-gRNA
FEATURES             Location/Qualifiers
     source          1..3567
                     /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"
     misc_RNA        955..1030
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1086..1106)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1086..1104)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1125..1141)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1125..1141)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1138..1160)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1149..1165
                     /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(1173..1179,1180..1197,1198..1203))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1218..1239
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1356..1373)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1527..2115)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1607..1626)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2252..3046)
                     /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(2252..3046)
                     /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(2363..2382)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2973..2992
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3081..3438)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3095..3230
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        3247..3438
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     3395..3415
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3440..3544)
                     /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 tgggtacctt
      661 taattctagt actatgcatc gtctcattgt ctgcagtatt tagcatgccc cacccatctg
      721 caaggcattc tggatagtgt caaaacagcc ggaaatcaag tccgtttatc tcaaacttta
      781 gcattttggg aataaatgat atttgctatg ctggttaaat tagattttag ttaaatttcc
      841 tgctgaagct ctagtacgat aagcaacttg acctaagtgt aaagttgaga cttccttcag
      901 gtttatatag cttgtgcgcc gcttgggtac ctcggtacac ggaccaccag cggcgtttta
      961 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     1021 gagtcggtgc tttttttctc ggactgatga cctgagacgc cgcggtggag ctccagcttt
     1081 tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct
     1141 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     1201 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     1261 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     1321 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     1381 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     1441 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     1501 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1561 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1621 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     1681 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     1741 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     1801 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     1861 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     1921 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     1981 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2041 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2101 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     2161 gaaaactcac gttaagggat tttggtcatg agcggataca tatttgaatg tatttagaaa
     2221 aataaacaaa taggggttcc gcgcatcatg atcagaagaa ctcgtcaaga aggcgataga
     2281 aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc
     2341 attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt
     2401 ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga
     2461 tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg ggcatgctcg
     2521 ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat
     2581 cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt
     2641 ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca
     2701 tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt
     2761 cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag
     2821 gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcttgcagt tcattcaggg
     2881 caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca
     2941 cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca
     3001 cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga aacgatcctc
     3061 atcctgtctc ttgatcgatc tttgcaaaag cctaggcctt caaaaaagcc tcctcactac
     3121 ttctggaata gctcagaggc cgaggcggcc tcggcctctg cataaataaa aaaaattagt
     3181 cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt
     3241 aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct
     3301 ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat gctttgcata
     3361 cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac attccacagc
     3421 tggttctttc cgcctcagga ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     3481 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     3541 cgcgcacatt tccccgaaaa gtgccac
//
