LOCUS       sequence-465448-        5231 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Generates GINS4 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 5231)
  TITLE     Chen knock-in cell line plasmids
REFERENCE   2  (bases 1 to 5231)
  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_GINS4_dTAG
FEATURES             Location/Qualifiers
     source          1..5231
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(145..468)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     150..169
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     363..385
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     377..394
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     378..394
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    395..451
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(464..480)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(464..480)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(477..499)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    488..504
                     /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(512..518,519..536,537..542))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    557..578
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(695..712)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(866..1454)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(946..965)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1625..2416,2417..2485))
                     /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     2248..2267
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2486..2590)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2658..2676
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2714..2736)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3567..3965)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(3966..4022)
                     /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(4026..4052)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(4056..4082)
                     /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(4056..4082)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(4092..4412)
                     /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"
ORIGIN
        1 attgaggcca ggagtttgac accagcctac gcaacatggc aagattctgt agacggtcac
       61 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
      121 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
      181 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca
      241 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
      301 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
      361 ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta cccggggatc
      421 ctctagagtc gacctgcagg catgcaagct tggcgtaatc atggtcatag ctgtttcctg
      481 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
      541 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
      601 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
      661 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
      721 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
      781 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      841 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      901 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      961 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1021 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1081 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1141 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1201 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1261 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     1321 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1381 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1441 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1501 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     1561 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     1621 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1681 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     1741 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     1801 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     1861 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     1921 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     1981 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2041 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     2101 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     2161 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     2221 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     2281 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     2341 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     2401 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     2461 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     2521 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     2581 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     2641 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     2701 atgacggtga aaacctctga cacatgcagc tcccggtctg ctgctccgcc aagtgtgcag
     2761 aacaaacagt acaatggccc catctactga cacgggctaa aacagcagac aggacatgca
     2821 gcaacacaag caatcctgcc tcgagctccg cttcacgccc gtgacccaca gcactgctga
     2881 ggaatgtctg atgcctagta aactacttta ttattgtcct caggcacaac gtgatacgag
     2941 gggtttctag tattttctct acggttccag gtcttcattc ttgtttacgc ctagtgtgtt
     3001 ttttttggaa atggagtctc gctctgtcgc ccaggctaga gtgcaatggc acgatcttgg
     3061 ctcactgcaa gctccacctc ccgggttcac accattctcc tgcctcagcc tcctgagtag
     3121 atgggcccgc caccatgcct ggctaatttt ttgtattttt agtagagatg ggtttagtag
     3181 agatggggtt tagtagccag gatggtcttg atctcctgac ctcatgagtt tatgcctagt
     3241 ttgttttttt tttgtttttt gttttttttt ttgagacaaa gtctcactgt cacccaggct
     3301 ggagtgcaat ggcacgacct cggctcactg caacctcctg ctcccaggtt caagcgattc
     3361 tcctgccccg gcctcctgag tagctgggat tacaggtgca caccaccaac acccggctaa
     3421 ttattttgta tttttagtaa agatggggtt tcaccatgtt ggtcggttgg tctcgaactc
     3481 ctgacctcat gatccgcccg cctcggcctc ccaaagtgct gggattacag gcttgagtca
     3541 ccatgcttgg ctgtttatgc gtagtttcag ccctcccaca cataaccaga gggcagcaat
     3601 tcacgaatcc caactgccgt cggctgtcca tcactgtcct tcactatggc tttgatccca
     3661 ggatgcagat cgagaagcac ctgtcggcac cgtccgcagg ggctcaagat gcccctgttc
     3721 tcatttccga tcgcgacgat acaagtcagg ttgccagctg ccgcagcagc agcagtgccc
     3781 agcaccacga gttctgcaca aggtccccca gtaaaatgat atacattgac accagtgaag
     3841 atgcggccgt cgctagagag agctgcgctg gcgacgctgt agtcttcaga gatggggatg
     3901 ctgttgattg tagccgttgc tctttcaatg agggtggatt cttcttgaga caaaggcttg
     3961 gccataggtc cagggttctc ctccacgtct ccagcctgct tcagcaggct gaagttagta
     4021 gcgcctgcat agtccgggac atcatacgga tagccggcgt agtcgggcac gtcgtagggg
     4081 tagcccgagc cttccagttt tagaagctcc acatcgaaga cgagagtggc atgtggtggg
     4141 atgatgcctg ggtgcccagt ggcaccatag gcataatctg gagatatagt cagtttggct
     4201 ctctgaccca cactcatctg ggcaacccct tcttcccagc ctcggatcac ctcctgcttg
     4261 cctagcataa acttaaaggg cttgtttctg tcccgggagg aatcaacttt ctttccatct
     4321 tcaagcatcc cggtgtagtg caccacgcag gtctggccgc gcttggggaa ggtgcgcccg
     4381 tctcctgggg agatggtttc cacctgcact ccagatccgc cgccacccga cccaccaccg
     4441 cccgagctac cgccaccaat tagctggaca gctcctgaag ctaccagagg tgcaatggtt
     4501 ttgtatcgga tcaagtgctg tgagcccttc tccaggtcaa tcacgtagtc cctgaaaaag
     4561 agggaaacac atttgcctgc agagctcggc tggccctcca tcgggaggaa tgtgcccact
     4621 gtgaaagatg cacgccactc acctctgctc atctgtgtct ggttctacca gtatgttttc
     4681 ttgtcgttct ctcactctca gaaacacgta agaatctaga tctggtttgg gaactgccag
     4741 gaaaacaaca atgtgattag gaaatcctcg gccaacaaag tcaacaaagt aaaagttgct
     4801 gggaagaatg gtatggcaca ctcagggttt agataggagc ccagcccatt cctactcttc
     4861 aaagatgcag acagagtggc tgaccaggga ggaaaccgga cacgcattgg gccttttgtt
     4921 ttcctcaggc agcctcagca aaatgaccaa ttcagggttc cacaagtctg gtatgggtct
     4981 gacttgctcc tggtgttatg ggctacactg tgtccctgca caattcatac gctgaagccc
     5041 taacccccag tatttcagcg tgggattgta ttcggaaaca gggcttttaa agaggcaatt
     5101 aagtgaaagc gaggccaagt agtgtgggcc ctaatccaat ctgactggtg tcttataaga
     5161 agagaatagg ccaggcacag tggcttatgc ctgtgatctc agcattttga gaggctgagg
     5221 ctggaggatc a
//
