LOCUS       sequence-321813-        4809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  homologous donor template for endogenous tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4809)
  AUTHORS   Gu B, Comerci CJ, McCarthy DG, Saurabh S, Moerner WE, Wysocka J
  TITLE     Opposing Effects of Cohesin and Transcription on CTCF Organization 
            Revealed by Super-resolution Imaging.
  JOURNAL   Mol Cell. 2020 Nov 19;80(4):699-711.e7. doi: 
            10.1016/j.molcel.2020.10.001. Epub 2020 Oct 21.
  PUBMED    33091336
REFERENCE   2  (bases 1 to 4809)
  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"; doi: 
            "10.1016/j.molcel.2020.10.001"; journalName: "Mol Cell"; date: 
            "2020-11-19- 19"; volume: "80"; issue: "4"; pages: "699-711.e7"
COMMENT     Sequence Label: pGEMT-halo-HA-p2a-neoR
FEATURES             Location/Qualifiers
     source          1..4809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(17..34)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(17..33)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(26..48)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      174..629
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(261..280)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     471..492
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        707..811
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(812..880,881..1672)
                     /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(1030..1049)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1843..2431
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2332..2351
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2585..2602
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2719..2740
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2755..2760,2761..2778,2779..2785)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2793..2809
                     /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     2798..2820
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2817..2833
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2817..2833
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2851..2869
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2851..2868
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(2937..3728)
                     /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="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3048..3067)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3658..3677
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3735..3791)
                     /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             complement(3801..3827)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3828..4712)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEIS"
     CDS             complement(4710..4736)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      4761..4770
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(join(4800..4809,1..10))
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(join(4801..4809,1..10))
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
       61 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
      121 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc
      181 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      241 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
      301 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
      361 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
      421 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
      481 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
      541 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
      601 ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg
      661 tgcggtattt cacaccgcat caggtggcac ttttcgggga aatgtgcgcg gaacccctat
      721 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
      781 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
      841 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
      901 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
      961 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     1021 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     1081 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     1141 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     1201 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     1261 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     1321 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     1381 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     1441 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     1501 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     1561 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     1621 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     1681 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     1741 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     1801 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     1861 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     1921 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     1981 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2041 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2101 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2161 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2221 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     2281 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2341 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     2401 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     2461 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     2521 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     2581 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     2641 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     2701 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     2761 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     2821 aaacagctat gaccatgatt acgccaagct atttaggtga cactatagaa tactcaagct
     2881 atgcatccaa cgcgttggga gctctcccat atggtcgacc tgcaggcggc cgcctgtcag
     2941 aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg
     3001 taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta
     3061 gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca
     3121 gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg
     3181 agatcatcgc cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc
     3241 ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt
     3301 gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta
     3361 tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat
     3421 gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg
     3481 acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct
     3541 gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg
     3601 cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc
     3661 cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct
     3721 tgttcaataa gcttaggtcc agggttctcc tccacgtctc cagcctgctt cagcaggctg
     3781 aagttagtag ctccgcttcc ggcgtagtct ggcacgtcat atgggtagga aatctccaga
     3841 gtagacagcc agcgcgcgat ctcgctgccg atcaggtccg ggttgtcttc ttgcagcaga
     3901 ttcagacccg ggccgatgtc cacagccttg cagttaggca ggcttttggc caggcgagcg
     3961 gcttcggccg gtgggatcag aacgcctggg gtgccccaga acagcagctt cgggacaggg
     4021 gactggtgca gccagtccat gtattcttcg accagcgcga cgatgttcgc tggctcaccg
     4081 gcgattggca gctcgtttgg gaagcgccac agtggctcgc ggtcaacagg attcaggaac
     4141 ggctcgcggt aatggtccat ctcgacttca gtcagcgggc ggacgacacc catcggcagc
     4201 gtaccctcga taaaaacgtt ctgatcgatg atcagcttgc ggccgacgtc ggtggtgcgg
     4261 aaggcctgga aggtctcgcg ggcaaattct ggccattcgt cccaggtcgg gatagggcgg
     4321 atgaactcca taaatgcaat acctttgacg cgctctggat tgcgcttggc ccagtggaaa
     4381 cccagagcgg agccccagtc gtgaatgacc aggacgacct cttccagacc cagggcttcg
     4441 atgaaggcat ccatgaagcg gacgtggtcg tcgaagaaat aacccaggtc tggtttgtcg
     4501 gatttgccca taccgatcag gtctggagca atgcagcgat gggtcggtgc aacatgcggg
     4561 atgatgttgc gccacacgta ggaggaggtc gggttaccgt gcaggaacag cacaggggtg
     4621 ccatcgcgcg gaccaacatc gacgtagtgc atgcgctcgc ccaggacttc cacataatgg
     4681 gggtcgaatg gaaagccagt accgatttcg gcgtagtctg gcacgtcata tgggtaggca
     4741 ccggctccag catcccgcgg ccatggcggc cgggagcatg cgacgtcggg cccaattcgc
     4801 cctatagtg
//
