LOCUS       sequence-304502-        5219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Replace donor vector for cherry insertion using the human Polb guide
            RNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5219)
  AUTHORS   Danner E, Lebedin M, de la Rosa K, Kuhn R
  TITLE     A homology independent sequence replacement strategy in human cells 
            using a CRISPR nuclease.
  JOURNAL   Open Biol. 2021 Jan;11(1):200283. doi: 10.1098/rsob.200283. Epub 
            2021 Jan 27.
  PUBMED    33499763
REFERENCE   2  (bases 1 to 5219)
  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: "Open 
            Biol."; date: "2021-01"; volume: "11(1):200283. doi"; pages: " 
            10.1098/rsob.200283. Epub 2021 Jan 27"
COMMENT     Sequence Label: pMC_cherry donor_Polb target (pE038)
FEATURES             Location/Qualifiers
     source          1..5219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(693..710)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    731..764
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             911..922
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             914..967
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             974..1684
                     /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(1119..1137)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1709..1726)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1715..1826
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(2084..2878)
                     /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(2084..2878)
                     /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(2195..2214)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2805..2824
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(3242..3259)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3412..3498
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3590..3617
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      3751..4339
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4240..4259
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4493..4510
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4525..4665
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4591..4610)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4751..4773
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tcttctctca tccgccaaaa cagccaagct ggagaccgtt tgacattacc ctgttatccc
       61 tagatacatt accctgttat cccagatgac ataccctgtt atccctagat gacattaccc
      121 tgttatccca gatgacatta ccctgttatc cctagataca ttaccctgtt atcccagatg
      181 acataccctg ttatccctag atgacattac cctgttatcc cagatgacat taccctgtta
      241 tccctagata cattaccctg ttatcccaga tgacataccc tgttatccct agatgacatt
      301 accctgttat cccagatgac attaccctgt tatccctaga tacattaccc tgttatccca
      361 gatgacatac cctgttatcc ctagatgaca ttaccctgtt atcccagatg acattaccct
      421 gttatcccta gatacattac cctgttatcc cagatgacat accctgttat ccctagatga
      481 cattaccctg ttatcccaga tgacattacc ctgttatccc tagatacatt accctgttat
      541 cccagatgac ataccctgtt atccctagat gacattaccc tgttatccca gatgacatta
      601 ccctgttatc cctagataca ttaccctgtt atcccagatg acataccctg ttatccctag
      661 atgacattac cctgttatcc cagataaact caatgatgat gatgatgatg gtcgagactc
      721 agcggccgcg gtgccagggc gtgcccttgg gctccccggg cgcgactagt gaattcagat
      781 ctgatatctc tagaagtcct gggtgaccca cacaaaatta gtcttttagc agactggtat
      841 gtttccaata agatcattta agtcctcaaa gcattcctaa atcattgtta gacttttttt
      901 tttcttaaag attgagggca gaggaagtct gctaacatgc ggtgacgtgg aggagaatcc
      961 cggccctgct agcatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     1021 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     1081 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     1141 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     1201 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     1261 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     1321 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     1381 agacggcccc gtaatgcaga agaaaaccat gggctgggag gcctcctccg agcggatgta
     1441 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     1501 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     1561 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     1621 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     1681 gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     1741 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1801 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     1861 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg
     1921 gggatccctg caggagctcg tcgacccatg ggggcccgcc ccaactgggg taacctttga
     1981 gttctctcag ttgggggtaa tcagcatcat gatgtggtac cacatcatga tgctgattat
     2041 aagaatgcgg ccgccacact ctagtggatc tcgagttaat aattcagaag aactcgtcaa
     2101 gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga
     2161 agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt
     2221 cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat
     2281 tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt
     2341 cgggcatgct cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt
     2401 cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc
     2461 gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca
     2521 ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct
     2581 gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca
     2641 cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcttgca
     2701 gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg
     2761 acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga
     2821 atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc
     2881 gaaacgatcc tcatcctgtc tcttgatcag agcttgatcc cctgcgccat cagatccttg
     2941 gcggcaagaa agccatccag tttactttgc agggcttccc aaccttacca gagggcgccc
     3001 cagctggcaa ttccggttcg cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg
     3061 taagcccact gcaagctacc tgctttctct ttgcgcttgc gttttccctt gtccagatag
     3121 cccagtagct gacattcatc cggggtcagc accgtttctg cggactggct ttctacgcgc
     3181 tcgagggggg gccaaacggt ctccagcttg gctgttttgg cggatgagag aagattttca
     3241 gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg
     3301 gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg
     3361 ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa
     3421 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     3481 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
     3541 gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc
     3601 ctgacggatg gcctttttgc gtttctacaa actcttttgt ttatttttct aaatacattc
     3661 aaatatgtat ccgctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3721 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3781 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3841 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     3901 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3961 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4021 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4081 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4141 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4201 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4261 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4321 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4381 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4441 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4501 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4561 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4621 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4681 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4741 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4801 aggcagcaga tcaattcgcg cgcgaaggcg aagcggcatg cataatgtgc ctgtcaaatg
     4861 gacgaagcag ggattctgca aaccctatgc tactccgtca agccgtcaat tgtctgattc
     4921 gttaccaatt atgacaactt gacggctaca tcattcactt tttcttcaca accggcacgg
     4981 aactcgctcg ggctggcccc ggtgcatttt ttaaataccc gcgagaaata gagttgatcg
     5041 tcaaaaccaa cattgcgacc gacggtggcg ataggcatcc gggtggtgct caaaagcagc
     5101 ttcgcctggc tgatacgttg gtcctcgcgc cagcttaaga cgctaatccc taactgctgg
     5161 cggaaaagat gtgacagacg cgacggcgac aagcaaacat gctgtgcgac gctggcgat
//
