LOCUS       sequence-321810-        4256 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 4256)
  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 4256)
  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-v5-dL5-p2a-blastR
FEATURES             Location/Qualifiers
     source          1..4256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(28..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(138..159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     350..369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     582..604
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     596..613
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     597..613
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     620..639
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        620..638
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      669..678
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             703..744
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             745..1464
                     /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"
     CDS             1474..1530
                     /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             1537..1935
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(2017..2035)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2018..2035)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2053..2069)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2053..2069)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2066..2088)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2077..2093
                     /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(2101..2107,2108..2125,2126..2131))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2146..2167
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2284..2301)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2455..3043)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2535..2554)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3214..4005,4006..4074))
                     /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     3837..3856
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4075..4179)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
      121 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa
      481 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
      541 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
      601 aacgacggcc agtgaattgt aatacgactc actatagggc gaattgggcc cgacgtcgca
      661 tgctcccggc cgccatggcc gcgggatgct ggagccggtg ccggtaagcc gattccaaac
      721 ccactgctgg gactggatag cacacaggcc gtcgttaccc aagaacctag tgttaccgtt
      781 agcccaggtg gtactgttat acttacttgt ggaagtggta cgggtgccgt cacatctggt
      841 cattatgcaa attggtttca acaaaaacca ggacaagctc caagagcttt gatttttgat
      901 actgataaga agtattcttg gaccccaggt agattttctg gatctttgct gggagcaaag
      961 gcagctttga caatatcaga tgctcagcct gaggacgaag ccgagtatta ctgttctctt
     1021 agcgacgtgg atggctactt gtttggcggt ggaacacaac tgacggttct gtccggtggt
     1081 ggcggctctg gtggcggtgg cagcggcggt ggtggttccg gaggcggcgg ttctcaggct
     1141 gtggtgactc aggagccgtc agtgactgtg tccccaggag ggacagtcat tctcacttgt
     1201 ggctccggca ctggagctgt caccagtggt cattatgcca actggttcca gcagaagcct
     1261 ggccaagccc ccagggcact tatatttgac accgacaaga agtattcctg gacccctggc
     1321 cgattctcag gctccctcct tggggccaag gctgccctga ccatctcgga tgcgcagcct
     1381 gaagatgagg ctgagtatta ctgttcgctc tccgacgttg acggttatct gttcggagga
     1441 ggcacccagc tgaccgtcct ctccggaagc ggagctacta acttcagcct gctgaagcag
     1501 gctggagacg tggaggagaa ccctggacct aagcttatgg ccaagccttt gtctcaagaa
     1561 gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac
     1621 tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta
     1681 tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg
     1741 gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc
     1801 ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg
     1861 aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat
     1921 gtgtgggagg gctaacaggc ggccgcacta gtgcggccgc ctgcaggtcg accatatggg
     1981 agagctccca acgcgttgga tgcatagctt gagtattcta tagtgtcacc taaatagctt
     2041 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     2101 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     2161 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     2221 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     2281 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     2341 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     2401 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     2461 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     2521 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     2581 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2641 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2701 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2761 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2821 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     2881 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2941 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3001 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3061 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3121 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3181 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3241 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3301 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     3361 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3421 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3481 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3541 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3601 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3661 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3721 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3781 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     3841 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3901 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3961 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4021 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4081 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4141 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     4201 acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagg
//
