LOCUS       sequence-189990-        6246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HMEJ donor for fusing a p2A-mCherry reporter to mouse Cdx2. AAV 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6246)
  AUTHORS   Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H, Shen X, Wei Y, Huang Z, 
            Ying W, Wang Y, Nie YH, Zhang CC, Li S, Cheng L, Wang Q, Wu Y, Huang
            P, Sun Q, Shi L, Yang H
  TITLE     Homology-mediated end joining-based targeted integration using 
            CRISPR/Cas9.
  JOURNAL   Cell Res. 2017 May 19. doi: 10.1038/cr.2017.76.
  PUBMED    28524166
REFERENCE   2  (bases 1 to 6246)
  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: "Cell Res. 
            2017 May 19. doi: 10.1038/cr.2017.76."
COMMENT     Sequence Label: AAV_Cdx2 HMEJ donor
FEATURES             Location/Qualifiers
     source          1..6246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     371..388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    505..526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(541..546,547..564,565..571)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..595
                     /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)."
     primer_bind     584..606
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     603..619
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     603..619
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(1192..1203)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1467..1523
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1524..2234
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1669..1687)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1937..1956
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3077..3096)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3078..3096)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3103..3120)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3103..3119)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3112..3134)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3260..3688
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3347..3366)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3557..3578
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             4032..4826
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4086..4105)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4696..4715
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(4844..4912,4913..5704)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5062..5081)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5875..6246,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      541 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      601 cacaggaaac agctatgacc atgattacgc caagcttggt accagaccac gggaggggtc
      661 actgggtgat tgcaggggac acccagtgcc tctggcttct gtggatatat atgaatgtat
      721 acagacatac acatatcaaa gatttaaaaa tccttgaact ttttttttca ttaacacaaa
      781 agaggcccta cttgcacacc aacactttct gtagtctagg gaaaagggaa ggaacaaaca
      841 agagaaattc atagccagga agttttggac aaagtttcat tgtctgtgcc aaggtgaaga
      901 acatcgcagt accaatgagc tgtgggagcc caagggtgac ccacgaggca cactgagctg
      961 gccaaccatt tgtgtaggga atagctttcg tcatggttcc gttccctggt tctgaggttc
     1021 tgttgctagt aaagggtttt ttgtttgttt gttttttgtt ttttgaaagc aacttgggga
     1081 gggggcaggt aagtaaagag gacgaggctg gcctggcttt gggaacattt cccaaactca
     1141 gtgaaagaag gacgctcccg gggtggcgcc atttctctgt tcttatcttc ttctaccttc
     1201 tattttgtca tctctaggtt aaaatttggt ttcagaaccg cagagccaag gagaggaaaa
     1261 tcaagaagaa gcagcagcag caacagcagc agcagcaaca acagcctcca cagccgccgc
     1321 cacaaccttc ccagcctcag ccgggtgccc tgcggagcgt gcccgagccc ttgagtcctg
     1381 tgacctcctt gcaaggctca gtgcctggtt ctgtccctgg ggttctgggg ccagctggag
     1441 gggttttaaa ctccactgtc acccaggcca ctaacttctc cctgttgaaa caagcagggg
     1501 atgtcgaaga gaatcccggg ccaatggtga gcaagggcga ggaggataac atggccatca
     1561 tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg
     1621 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
     1681 tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg
     1741 gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc
     1801 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga
     1861 cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca
     1921 acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg
     1981 agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga
     2041 aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc
     2101 agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact
     2161 acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg
     2221 agctgtacaa gtaagacccc tcccgtggtc tgaagcggcg gcggcacagc aatcccaggc
     2281 tgagccatga ggagtatgga cgctgcgaga atcctcagaa gagattcctc tcctcctacc
     2341 cacgaacagc atctactgat ggagattgag gacagaagat gagtggaatt atggacctca
     2401 ggggaagaca tggtttagat tttttttttt ctttttaact tttcccattc cgactcttcc
     2461 tgccagcaac gacaaacgaa gtgattcctg gggcttcttc gttcatgctc tttgccagga
     2521 ctgactaccg acatgaagct atcagcctct tttgccccag ctctttgcct ctctgtattt
     2581 ctgtgtggag ctgaggagag agtgagactg gatggggtgg gggtagcaat acttgagcca
     2641 aggtggctgt ttcctgctga ctgctttctg agaaccagct ggccgtcctg cctccgggcc
     2701 agggactatt caaactacag gagccagagg cagctaagat agctggactg accgaagtct
     2761 gcagaacctc ccccaccagg tggtctgggc tttcttctcc acaaatcagg aaggggtggt
     2821 gggttcaggg gctgcggtga gagggggttg gttagccaac gccaggcccc tgcgacaagg
     2881 gcttgtttag aaagcctgtc accagagctg ctgtaggcgg aatgtatgtc tgtgttgtaa
     2941 atgccagagc caacctggac ttcctgtccc ttccctcgtc tttggctgaa gaagaccgga
     3001 attgtttgct gctgttcgag tcactgatct gtgtacccag tgacccctcc cgtggtcttc
     3061 tagagggccc aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca
     3121 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     3181 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     3241 cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3301 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3361 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3421 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3481 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3541 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3601 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3661 ctgatttaac aaaaatttaa cgcgaatttt aacaaaattc agggcgcaag ggctgctaaa
     3721 ggaagcggaa cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca
     3781 gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca
     3841 gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat
     3901 tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt
     3961 tcttgccgcc aaggatctga tggcgcaggg gatcaagatc tgatcaagag acaggatgag
     4021 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4081 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4141 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4201 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4261 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4321 tgccggggca ggatctcctg tcatcccacc ttgctcctgc cgagaaagta tccatcatgg
     4381 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4441 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4501 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4561 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4621 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4681 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4741 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4801 atcgccttct tgacgagttc ttctgaattg aaaaaggaag agtatgagta ttcaacattt
     4861 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4921 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     4981 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5041 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5101 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5161 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5221 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5281 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5341 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5401 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5461 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5521 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     5581 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     5641 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     5701 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     5761 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5821 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5881 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5941 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6001 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6061 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6121 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6181 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6241 cgaact
//
