LOCUS       sequence-135361-        7226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for 3' FLAG tag of human ZNF146.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7226)
  TITLE     Mendenhall CRISPR tagging plasmids
REFERENCE   2  (bases 1 to 7226)
  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"
COMMENT     Sequence Label: pFETCh_ZNF146
FEATURES             Location/Qualifiers
     source          1..7226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             253..261
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             909..932
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             936..959
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             963..986
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1002..1058
                     /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             1071..1865
                     /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(1125..1144)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1735..1754
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2728..2744)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2737..2759)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(2963..3778)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3686..3705
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3770..3789
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4158..4746
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4647..4666
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4900..4917
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5217..5238
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5253..5258,5259..5276,5277..5283)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5291..5307
                     /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     5296..5318
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5315..5331
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5315..5331
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5508..5710
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5661..5681
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5707..5731
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5807..6937
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDAL
                     LQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
ORIGIN
        1 cacacctcag ccagcagaga atttacagtg gggaaaaccc ctttgcctgt aaggtatgtg
       61 gaaaagtctt cagccacaaa tcaaacctca ctgagcatga gcattttcac acgagagaga
      121 aaccttttga atgtaacgag tgtggaaaag cctttagcca aaagcagtat gtcattaaac
      181 atcagaacac ccatactggc gagaagcttt tcgaatgtaa tgaatgtgga aaatcattta
      241 gccagaagga aaacctcctt acgcaccaga aaattcacac tggagaaaaa ccttttgagt
      301 gtaaagattg cgggaaagct ttcattcaga agtcaaacct catcagacac cagagaactc
      361 acacaggaga gaagcccttt gtatgtaagg agtgtggaaa aaccttcagt ggcaaatcca
      421 accttactga gcatgagaaa atccatattg gagagaagcc ttttaaatgt agtgaatgtg
      481 gaacagcctt tggccagaag aagtacctca taaaacatca gaacattcac actggagaga
      541 aaccctatga atgtaacgaa tgtggaaaag ccttctctca gcgaacatca cttattgtac
      601 atgtgaggat tcattcaggt gataaacctt acgaatgcaa tgtttgtgga aaagccttct
      661 ctcagagctc atctctcact gtgcatgtga gaagccatac aggggagaag ccctatggtt
      721 gtaatgaatg tgggaaagct ttctctcagt tctcaaccct tgctctgcat ttgagaatac
      781 acacaggtaa gaagccttat cagtgcagtg aatgtgggaa agctttcagc cagaagtcac
      841 accacattag acaccagaaa attcatactc acgggagcgg aggaggttcc ggtggaggtg
      901 gttctggaga ttacaaggat gacgacgata agggcgatta caaggatgac gacgataagg
      961 gagattacaa ggatgacgac gataaggttt caggaagcgg agctactaac ttcagcctgc
     1021 tgaagcaggc tggagacgtg gaggagaacc ctggacctgg atcgtttcgc atgattgaac
     1081 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     1141 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     1201 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     1261 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     1321 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     1381 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     1441 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     1501 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     1561 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     1621 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     1681 ctggattcat cgactgtggc cggctgggtg tggccgaccg ctatcaggac atagcgttgg
     1741 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     1801 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     1861 tctgattcga acatcaaagc cttgaaagtg ggaaagcttt cattagaaat ttgcacctca
     1921 tcatgcccca gaaataatcc ttctgaagca aagcaccacg aatgaggtta actttaacaa
     1981 gtactaaaaa cttaagggac accagaaaat ttgtactgaa gagaaagaca tgcatatgat
     2041 taaaaccctg tgtccaacag agaaacctgc agcagagata atggtgaaag tttaggcaca
     2101 ttttcactaa aagtgggaac agaaaatgga ggcctgtttt ttattgctac caccatagat
     2161 ctggagatct tcgccagtaa caagaaaaat taagttgtaa atattggaaa ggaacagaca
     2221 aaaatagatg acatggtcat ctacaactaa tattaagact cctgtggtat tgattgagca
     2281 gagcagaaga gatttcaaaa aaagacctat gcatttatta gaatttggaa tatgatacaa
     2341 gtggcatcag gaaatgagga aataatggaa ctattttttt taagtggagg tagtttgttc
     2401 tccaaggggg aaaaatagac catgaactat acacaaaagt gaatccagag attaaaaaca
     2461 gaaatatgga aaagctatgt actaaaatgc atatgacctt gggaataggg aaacattcct
     2521 tggaacaatt tgagaaatac ttagcggttc tacttatgag tatgaatgct caggaaatac
     2581 gcactaggat atttactgtg gcttgatttg tagtagccaa aaattagaaa caactgagaa
     2641 agcccatcac gaacaatatg ggaaattctt atgtacttgg aaagtcctct ccactgcctg
     2701 tagcctccaa ttcactggcc gtcgttcact ggccgtcgtt ttacaacgtc gtgactggga
     2761 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     2821 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     2881 atgcgattta ttcaacaaag ccgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt
     2941 acaaccaatt aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt
     3001 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     3061 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     3121 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     3181 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctta tgcatttctt
     3241 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     3301 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     3361 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3421 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     3481 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3541 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3601 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     3661 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3721 ccatgttgga atttaatcgc ggcttcgagc aagacgtttc ccgttgaata tggctcataa
     3781 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt
     3841 tatcttgtgc aatgtaacat cagagatttt gagacacaac gtggctttgt tgaataaatc
     3901 gaacttttgc tgagttgaag gatcagatca cgcatcttcc cgacaacgca gaccgttccg
     3961 tggcaaagca aaagttcaaa atcaccaact ggtccaccta caacaaagct ctcatcaacc
     4021 gtggctccct cactttctgg ctggatgatg gggcgattca ggcctggtat gagtcagcaa
     4081 caccttcttc acgaggcaga cctctcgacg gagttccact gagcgtcaga ccccgtagaa
     4141 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4201 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4261 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4321 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4381 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4441 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4501 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4561 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4621 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4681 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4741 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4801 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4861 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4921 gcggaagaag ctcgcacatt cagcagcgtt tttcagcgcg ttttcgatca acgtttcaat
     4981 gttggtatca acaccaggtt taactttgaa cttatcggca ctgacggtta ctgattttga
     5041 acttttgctt tgccacggaa cggtctgcgt tgtcgggaag atgcgtgatc tgatccttca
     5101 actcagcaaa agttcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     5161 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     5221 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     5281 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     5341 gccaagcttg catgcctgca ggtcgactct agaggatccc cgggtaccga gctcgaatta
     5401 gcttgcatgc ctgcaggtca attccctggc attatgccca gtacatgacc ttatgggact
     5461 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     5521 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcaa gtctccaccc
     5581 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     5641 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     5701 aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
     5761 cctccatagg agacaccggg accgatccag cctggggatc ccgaccatgg cttcgtaccc
     5821 ctgccatcaa cacgcgtctg cgttcgacca ggctgcgcgt tctcgcggcc atagcaaccg
     5881 acgtacggcg ttgcgccctc gccggcagca agaagccacg gaagtccgcc tggagcagaa
     5941 aatgcccacg ctactgcggg tttatataga cggtcctcac gggatgggga aaaccaccac
     6001 cacgcaactg ctggtggccc tgggttcgcg cgacgatatc gtctacgtac ccgagccgat
     6061 gacttactgg caggtgctgg gggcttccga gacaatcgcg aacatctaca ccacacaaca
     6121 ccgcctcgac cagggtgaga tatcggccgg ggacgcggcg gtggtaatga caagcgccca
     6181 gataacaatg ggcatgcctt atgccgtgac cgacgccgtt ctggctcctc atatcggggg
     6241 ggaggctggg agctcacatg ccccgccccc ggccctcacc ctcatcttcg accgccatcc
     6301 catcgccgcc ctcctgtgct acccggccgc gcgatacctt atgggcagca tgacccccca
     6361 ggccgtgctg gcgttcgtgg ccctcatccc gccgaccttg cccggcacaa acatcgtgtt
     6421 gggggccctt ccggaggaca gacacatcga ccgcctggcc aaacgccagc gccccggcga
     6481 gcggcttgac ctggctatgc tggccgcgat tcgccgcgtt tacgggctgc ttgccaatac
     6541 ggtgcggtat ctgcagggcg gcgggtcgtg gcgggaggat tggggacagc tttcggggac
     6601 ggccgtgccg ccccagggtg ccgagcccca gagcaacgcg ggcccacgac cccatatcgg
     6661 ggacacgtta tttaccctgt ttcgggcccc cgagttgctg gcccccaacg gcgacctgta
     6721 caacgtgttt gcctgggcct tggacgtctt ggccaaacgc ctccgtccca tgcacgtctt
     6781 tatcctggat tacgaccaat cgcccgccgg ctgccgggac gccctgctgc aacttacctc
     6841 cgggatggtc cagacccacg tcaccacccc cggctccata ccgacgatct gcgacctggc
     6901 gcgcacgttt gcccgggaga tgggggaggc taactgagga tcccacgtca ctattgtata
     6961 ctctatatta tactctatgt tatactctgt aatcctactc aataaacgtg tcacgcctgt
     7021 gaaaccgtac taagtctccc gtgtattctt atcaccatca ggtgacatcc tcgcccaggc
     7081 tgtcaatcat gccggtatcg attccagtag caccggcccc acgctgacaa cccactcttg
     7141 cagcgttagc agcgcccctc ttaacaagcc gacccccacc agcgtcgcgg ttactaacac
     7201 tcctctcccc gacctgcagc ccagct
//
