LOCUS       sequence-321811-        4254 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 4254)
  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 4254)
  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-blastR-p2a-v5-dL5
FEATURES             Location/Qualifiers
     source          1..4254
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(543..1334,1335..1403))
                     /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     1166..1185
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1404..1508)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1586..2041)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1723..1744)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1935..1954
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2167..2189
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2181..2198
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2182..2198
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2205..2224
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2205..2223
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2254..2263
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2273..2668
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             2678..2734
                     /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             2741..2782
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2783..3502
                     /codon_start=1
                     /product="fluorogen-activating protein composed of a dimer 
                     of the L5-MG single-chain antibody fragment (Szent-Gyorgyi 
                     et al., 2008)"
                     /label=FAP-alpha-2
                     /note="binds malachite green derivatives to produce red 
                     fluorescence"
                     /translation="QAVVTQEPSVTVSPGGTVILTCGSGTGAVTSGHYANWFQQKPGQA
                     PRALIFDTDKKYSWTPGRFSGSLLGAKAALTISDAQPEDEAEYYCSLSDVDGYLFGGGT
                     QLTVLSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSVTVSPGGTVILTCGSGTGAVTSGH
                     YANWFQQKPGQAPRALIFDTDKKYSWTPGRFSGSLLGAKAALTISDAQPEDEAEYYCSL
                     SDVDGYLFGGGTQLTVLS"
     promoter        complement(3600..3618)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3601..3618)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3636..3652)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3636..3652)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3649..3671)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3660..3676
                     /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(3684..3690,3691..3708,3709..3714))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3729..3750
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3867..3884)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(4038..4254,1..372))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4118..4137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
       61 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
      121 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
      181 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
      241 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
      301 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
      361 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
      421 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
      481 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
      541 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
      601 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
      661 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
      721 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
      781 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
      841 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
      901 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
      961 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     1021 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     1081 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     1141 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     1201 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     1261 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     1321 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     1381 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     1441 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     1501 gttccgcgca catttccccg aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg
     1561 cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg
     1621 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     1681 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     1741 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     1801 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     1861 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     1921 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     1981 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     2041 tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     2101 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     2161 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat
     2221 agggcgaatt gggcccgacg tcgcatgctc ccggccgcca tggccgcggg atatggccaa
     2281 gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat
     2341 ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt
     2401 cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg
     2461 cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa
     2521 caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg
     2581 gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt
     2641 gctgccctct ggttatgtgt gggagggcgg aagcggagct actaacttca gcctgctgaa
     2701 gcaggctgga gacgtggagg agaaccctgg acctaagctt ggtaagccga ttccaaaccc
     2761 actgctggga ctggatagca cacaggccgt cgttacccaa gaacctagtg ttaccgttag
     2821 cccaggtggt actgttatac ttacttgtgg aagtggtacg ggtgccgtca catctggtca
     2881 ttatgcaaat tggtttcaac aaaaaccagg acaagctcca agagctttga tttttgatac
     2941 tgataagaag tattcttgga ccccaggtag attttctgga tctttgctgg gagcaaaggc
     3001 agctttgaca atatcagatg ctcagcctga ggacgaagcc gagtattact gttctcttag
     3061 cgacgtggat ggctacttgt ttggcggtgg aacacaactg acggttctgt ccggtggtgg
     3121 cggctctggt ggcggtggca gcggcggtgg tggttccgga ggcggcggtt ctcaggctgt
     3181 ggtgactcag gagccgtcag tgactgtgtc cccaggaggg acagtcattc tcacttgtgg
     3241 ctccggcact ggagctgtca ccagtggtca ttatgccaac tggttccagc agaagcctgg
     3301 ccaagccccc agggcactta tatttgacac cgacaagaag tattcctgga cccctggccg
     3361 attctcaggc tccctccttg gggccaaggc tgccctgacc atctcggatg cgcagcctga
     3421 agatgaggct gagtattact gttcgctctc cgacgttgac ggttatctgt tcggaggagg
     3481 cacccagctg accgtcctct ccggaggagg tgggtctgga ggtggaggat ctcaggcggc
     3541 cgcctgcagg tcgaccatat gggagagctc ccaacgcgtt ggatgcatag cttgagtatt
     3601 ctatagtgtc acctaaatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3661 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3721 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3781 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3841 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3901 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3961 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4021 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4081 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4141 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4201 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctc
//
