LOCUS       sequence-310238-        7472 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector to introduce an Halotag cassette at the mouse Rad21
            locus using NEOMYCINE selection. Designed for using with sgRNA 
            CCACGGTTCCATATTATCTG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7472)
  AUTHORS   Nora EP, Caccianini L, Fudenberg G, So K, Kameswaran V, Nagle A, 
            Uebersohn A, Hajj B, Saux AL, Coulon A, Mirny LA, Pollard KS, Dahan 
            M, Bruneau BG
  TITLE     Molecular basis of CTCF binding polarity in genome folding.
  JOURNAL   Nat Commun. 2020 Nov 5;11(1):5612. doi: 10.1038/s41467-020-19283-x.
  PUBMED    33154377
REFERENCE   2  (bases 1 to 7472)
  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: "2020-11-5"; volume: "11(1):5612. doi"; pages: " 
            10.1038/s41467-020-19283-x"
COMMENT     Sequence Label: pEN313 - Rad21-Halo-Frt-PGK-EM7-NeoR-bpA-Frt
            t...
FEATURES             Location/Qualifiers
     source          1..7472
                     /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     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1604..2488
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     protein_bind    2499..2532
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     promoter        2596..3095
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2631..2654)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3028..3047
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3107..3154
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3173..3976
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3236..3255)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3846..3865
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4008..4025)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4014..4238
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4293..4326
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(4346..4362)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4346..4362)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(5284..5302)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5323..5339)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5323..5339)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5336..5358)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5347..5363
                     /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(5371..5377,5378..5395,5396..5401))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5416..5437
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5554..5571)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5725..6313)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5805..5824)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6484..7275,7276..7344))
                     /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     7107..7126
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7345..7449)
                     /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 gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtacctcagc
      661 agatgcttca tggtcttcag gtatgccagc acagtccaca aagagtcctt tcccagaaag
      721 caagtcctgt taaaccccga agcatgcctt ggtgggaatt tctccagggc tgcattgttt
      781 taaagcatct tagttacata catgaagtgt caagccaggg gtaaacctag agcatttagt
      841 ttatgtagcc atgattatcc taaaaaacct gatgcatggt gctgtgatgg ggagcgcagc
      901 tgcgtactgt cttgtttgat tagcaaaagc catgtgctaa gtcatagagt agcagaagtc
      961 agcaacctca gctagggttc cagatacagg tgaggctgga gcacccgtga cagttctccc
     1021 aagttccttc ctttctgcac atggggatgg atgatactta gggttggaag gttatcaggg
     1081 gccggtttga ttttggtttt gttttcactt taaaatctgc tgagtgtttg tttttgctaa
     1141 ctcacatctc tgttgtggcg ccttcactta cttcttgaac tctgtatgcc ttaagataat
     1201 atgctttaga taataaggcc tttaccttag tgactagcat gggaaccact tgcgtaacta
     1261 cacgaatgtc cagtcccagc tccagccaaa cggagctcac tttgaccagt gccaaaattg
     1321 catcttctgg ttactacttt gtgcgtgagt tacttgaaat catctgcttt gttttgttct
     1381 atttcagcga gctcttgcta aaactggagc agagtctatc agtttgcttg agctgtgtcg
     1441 aaacacaaac cgaaagcagg cagcagcaaa gttctacagc tttttggttc ttaagaagca
     1501 gcaagccatc gagctcacac aggaagagcc gtacagtgac atcattgcaa cccctggacc
     1561 acggttccat attatcctcg aggggggagc aggaggcgga tccgaaatcg gtactggctt
     1621 tccattcgac ccccattatg tggaagtcct gggcgagcgc atgcactacg tcgatgttgg
     1681 tccgcgcgat ggcacccctg tgctgttcct gcacggtaac ccgacctcct cctacgtgtg
     1741 gcgcaacatc atcccgcatg ttgcaccgac ccatcgctgc attgctccag acctgatcgg
     1801 tatgggcaaa tccgacaaac cagacctggg ttatttcttc gacgaccacg tccgcttcat
     1861 ggatgccttc atcgaagccc tgggtctgga agaggtcgtc ctggtcattc acgactgggg
     1921 ctccgctctg ggtttccact gggccaagcg caatccagag cgcgtcaaag gtattgcatt
     1981 tatggagttc atccgcccta tcccgacctg ggacgaatgg ccagaatttg cccgcgagac
     2041 cttccaggcc ttccgcacca ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt
     2101 tatcgagggt acgctgccga tgggtgtcgt ccgcccgctg actgaagtcg agatggacca
     2161 ttaccgcgag ccgttcctga atcctgttga ccgcgagcca ctgtggcgct tcccaaacga
     2221 gctgccaatc gccggtgagc cagcgaacat cgtcgcgctg gtcgaagaat acatggactg
     2281 gctgcaccag tcccctgtcc cgaagctgct gttctggggc accccaggcg ttctgatccc
     2341 accggccgaa gccgctcgcc tggccaaaag cctgcctaac tgcaaggctg tggacatcgg
     2401 cccgggtctg aatctgctgc aagaagacaa cccggacctg atcggcagcg agatcgcgcg
     2461 ctggctgtct actctggaga tttccggtta agaattccga agttcctatt ctctagaaag
     2521 tataggaact tcaggtctga agaggagttt acgtccagcc aagctagctt ggctgcaggt
     2581 cgtcgaaatt ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt
     2641 tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca
     2701 catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta
     2761 ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac
     2821 aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga
     2881 agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc
     2941 agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg
     3001 gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg
     3061 cgctgttctc ctcttcctca tctccgggcc tttcgacctg cagcctgttg acaattaatc
     3121 atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa ccatgggatc
     3181 ggccattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3241 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3301 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3361 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3421 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3481 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3541 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3601 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3661 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     3721 cggcgatgat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     3781 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     3841 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     3901 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     3961 tgacgagttc ttctgagggg atcaattctc tagagctcgc tgatcagcct cgactgtgcc
     4021 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4081 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4141 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4201 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     4261 ctggggctcg actagagctt gcggaaccct tcgaagttcc tattctctag aaagtatagg
     4321 aacttcatca gtcaggtaca taatggatcc actagttcta gacatatggg agctagatgt
     4381 gttcgagcta gtgataactc actagtacat acaaattgcc cccgtgtgca gggcaccaaa
     4441 accctttaag aaagttttta gatttctgtt tgtacaaaaa tctttgcctt ttctttcttc
     4501 tttttccccc cagtgtttct aattttgtca accatatttt taagggaaac tgcttatttg
     4561 ggttgggttt gtattcctgg agaaaacagt agcccaagaa cccagaagac ttttaacagt
     4621 tcagaacaga tgtgtgcaat attggtgcat gtaagaatat ggagtaacag tcaaaaggca
     4681 ccatttttaa tgttagtttt ccattactat gttgaaagga aaacctgcct aggaaaatgc
     4741 ctgacacttt aagaactgtg gtttgagtcc cttgacagga agagaaaaat gtcttcccat
     4801 cagtgaaacc aacggtctgg ttaaccactg tagtagggat agtgtgtgaa gcatcgtgct
     4861 tgtcttcttg atcatgttta aagtaaatcc acactgagca agaatttgtg aggttgtgct
     4921 gactgatctt cttcggctat tatgttctga aatcattccg tgagtcttag gacctctgat
     4981 gctatgcacc gcagctcaga acgatcagac gtctacagtg gcgtgactcc tcaggggctg
     5041 cagatcactc tcagaggtgt ttggatgttt gcttgaataa tgagatcatg gaagcagaca
     5101 ttcccctgcc tgctgaaggg cagaccaatg agagactacc ccagattgac ttcctttaag
     5161 caaacagtgc tgtaaaaact aatggcttct ctgatattta ttataaatgt tagtactcat
     5221 ctttttccaa ggctgcacac tcctgtagcg gccgccaccg cggtggagct ccagcttttg
     5281 ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt
     5341 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5401 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5461 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5521 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5581 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5641 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5701 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5761 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5821 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5881 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5941 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6001 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6061 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6121 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6181 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6241 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6301 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6361 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6421 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6481 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6541 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6601 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6661 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6721 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6781 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6841 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6901 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6961 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7021 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7081 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7141 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7201 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7261 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7321 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7381 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7441 ggttccgcgc acatttcccc gaaaagtgcc ac
//
