LOCUS       sequence-465447-        4874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Generates MCM2 dTAG C ternimal  knock-in cell lines as donor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4874)
  TITLE     Chen knock-in cell line plasmids
REFERENCE   2  (bases 1 to 4874)
  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: pUC19_MCM2_dTAG
FEATURES             Location/Qualifiers
     source          1..4874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     184..206
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     198..215
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     199..215
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    216..272
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(285..301)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(285..301)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(298..320)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    309..325
                     /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(333..339,340..357,358..363))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    378..399
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(516..533)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(687..1275)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(767..786)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1446..2237,2238..2306))
                     /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     2069..2088
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2307..2411)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2479..2497
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2535..2557)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3175..3573)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(3574..3630)
                     /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(3634..3660)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3664..3690)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             complement(3664..3690)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3700..4020)
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             complement(join(4840..4874,1..289))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     4845..4864
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
       61 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      121 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      181 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat
      241 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct
      301 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
      361 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
      421 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
      481 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
      541 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
      601 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
      661 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
      721 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
      781 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
      841 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
      901 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
      961 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     1021 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     1081 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     1141 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     1201 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     1261 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     1321 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     1381 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     1441 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     1501 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     1561 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     1621 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     1681 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     1741 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     1801 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     1861 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     1921 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     1981 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     2041 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     2101 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     2161 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     2221 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     2281 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     2341 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     2401 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     2461 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     2521 gatgacggtg aaaacctctg acacatgcag ctcccggaac cgaatgcaca aataagagag
     2581 tggggaaaca gacagggagc aatggcagct gataactgtg gcgagtggat aatccgcatg
     2641 actcacatcc cacccctgcg actgaggagc ccagaacgct gcgctggcac tgtcgtgccc
     2701 tcttccacca caggggcaca gaaacctgca ggagactgaa aacgattaca aacatcttca
     2761 accagctctc tggccaaagg cagcaaaagc agcctgaata cgcaactcac gccaagaggg
     2821 cagcagctct cctgacatcc atgtaagaag gctaacacct aaaccacacg caggcatcct
     2881 gaactcagca gctctgatcc aaggtactga gtggagacaa agcactcgga ggtggcaaga
     2941 tgttcagcaa ccaagtaaga cacactggca aggcatccca cccaaaggtg agaagcacaa
     3001 agcaggcttg gagaaacaaa cagtcatgcc aggtgcagcc agacatcctg ctataagccc
     3061 tgaccctagt accccgagtt catcaagtgc tctggttttg tgtccataaa gcacagaggg
     3121 cactgaccac cccaaaccag aatcccaagg aatcgttatg gatggcatag ggcctcagcc
     3181 ctcccacaca taaccagagg gcagcaattc acgaatccca actgccgtcg gctgtccatc
     3241 actgtccttc actatggctt tgatcccagg atgcagatcg agaagcacct gtcggcaccg
     3301 tccgcagggg ctcaagatgc ccctgttctc atttccgatc gcgacgatac aagtcaggtt
     3361 gccagctgcc gcagcagcag cagtgcccag caccacgagt tctgcacaag gtcccccagt
     3421 aaaatgatat acattgacac cagtgaagat gcggccgtcg ctagagagag ctgcgctggc
     3481 gacgctgtag tcttcagaga tggggatgct gttgattgta gccgttgctc tttcaatgag
     3541 ggtggattct tcttgagaca aaggcttggc cataggtcca gggttctcct ccacgtctcc
     3601 agcctgcttc agcaggctga agttagtagc gcctgcatag tccgggacat catacggata
     3661 gccggcgtag tcgggcacgt cgtaggggta gcccgagcct tccagtttta gaagctccac
     3721 atcgaagacg agagtggcat gtggtgggat gatgcctggg tgcccagtgg caccataggc
     3781 ataatctgga gatatagtca gtttggctct ctgacccaca ctcatctggg caaccccttc
     3841 ttcccagcct cggatcacct cctgcttgcc tagcataaac ttaaagggct tgtttctgtc
     3901 ccgggaggaa tcaactttct ttccatcttc aagcatcccg gtgtagtgca ccacgcaggt
     3961 ctggccgcgc ttggggaagg tgcgcccgtc tcctggggag atggtttcca cctgcactcc
     4021 agatccgccg ccacccgacc caccaccgcc cgagccaccg ccaccgaact gctgcaggat
     4081 cattttcctt ttcaggtcgt ggctgaactt gttcatcctg aagagctcac tgtcataaaa
     4141 tgcagagagg ttgtggatgt tgatctgacg agcctgcaag gaggaagtgt ccagtgagcc
     4201 atcagtggtg tgagaacagg ctgtttccag agcgccccag ttacagtgca ctttcagaca
     4261 ccatcacctc catttatggg aggaaaatat ccaggggtgg agaggccgat taaggggctt
     4321 caaatctctg ctgtacaagc tgtgcgtgta aactgagaca agttacttgg ctgccctgta
     4381 tctgtctcca cctctgcaca acaggaaggc agtaacagga ctcacctcac tcccctgctg
     4441 tcagactaca ggcacagaac tggactcaac agactgtaag cattcaataa atggctgtca
     4501 accaaggccc acccacttca tgacccatta tgatggagcc cccatcagga gactggcagc
     4561 tcccaagtca gcatacccca accctcacct cccttccggg cccatacctt atccaccaag
     4621 tccttctcag ggacctcaat agtgtcctgc tgggccccaa agcggttgcg ctgatatgtc
     4681 acctgctctg ccactaactg cttcagtatg aagagcaaca gctcattgtt gtcacgccgg
     4741 aatgaagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     4801 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     4861 tgtactgaga gtgc
//
