LOCUS       sequence-378107-        7270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor for targeted integration of 3xFLAG-2xSTREP tagged S-phase 
            specific probe (FUCCI-S) to the AAVS1 safe harbor locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7270)
  TITLE     S-phase specific probe
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7270)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: AAVS1_Puro_hPGK1_hGEM(1-110)_mNG_hSLBP(18-108)_FS
FEATURES             Location/Qualifiers
     source          1..7270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    320..1123
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    1130..1155
                     /label=SA
                     /note="splice acceptor site"
     CDS             1179..1232
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1236..1832
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     1729..1749
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1848..1969
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1859..1878)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1913..1932
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2042..2552
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2471..2489
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2571..2900
                     /codon_start=1
                     /product="degron consisting of residues 1-110 of human 
                     Geminin (Zielke and Edgar, 2015)"
                     /label=Gem1 (1-110)
                     /translation="MNPSMKQKQEEIKENIKNSSVPRRTLKMIQPSASGSLVGRENELS
                     AGLSKRKHRNDHLTSTTSSPGVIVPESSENKNLGGVTQESFDLMIKENPSSQYWKEVAE
                     KRRKAL"
     CDS             2913..3620
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     CDS             3936..4001
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(4011..4016,4017..4040,4041..4076,4077..4100)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     polyA_signal    4123..4347
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    4367..5203
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(5280..5297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5451..6039)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5531..5550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6210..7001,7002..7070))
                     /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     6833..6852
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7071..7175)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7243..7261
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagaa ttcgcccttt gctttctctg accagcattc tctcccctgg gcctgtgccg
      361 ctttctgtct gcagcttgtg gcctgggtca cctctacggc tggcccagat ccttccctgc
      421 cgcctccttc aggttccgtc ttcctccact ccctcttccc cttgctctct gctgtgttgc
      481 tgcccaagga tgctctttcc ggagcacttc cttctcggcg ctgcaccacg tgatgtcctc
      541 tgagcggatc ctccccgtgt ctgggtcctc tccgggcatc tctcctccct cacccaaccc
      601 catgccgtct tcactcgctg ggttcccttt tccttctcct tctggggcct gtgccatctc
      661 tcgtttctta ggatggcctt ctccgacgga tgtctccctt gcgtcccgcc tccccttctt
      721 gtaggcctgc atcatcaccg tttttctgga caaccccaaa gtaccccgtc tccctggctt
      781 tagccacctc tccatcctct tgctttcttt gcctggacac cccgttctcc tgtggattcg
      841 ggtcacctct cactcctttc atttgggcag ctcccctacc ccccttacct ctctagtctg
      901 tgctagctct tccagccccc tgtcatggca tcttccaggg gtccgagagc tcagctagtc
      961 ttcttcctcc aacccgggcc cctatgtcca cttcaggaca gcatgtttgc tgcctccagg
     1021 gatcctgtgt ccccgagctg ggaccacctt atattcccag ggccggttaa tgtggctctg
     1081 gttctgggta cttttatctg tcccctccac cccacagtgg ggcaagcttc tgacctcttc
     1141 tcttcctccc acagggcctc gagagatctg gcggcggaga gggcagagga agtcttctaa
     1201 catgcggtga cgtggaggag aatcccggcc ctagcaccga gtacaagccc acggtgcgcc
     1261 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     1321 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     1381 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     1441 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     1501 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     1561 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     1621 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     1681 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     1741 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     1801 cctggtgcat gacccgcaag cccggtgcct gatctagaca gacatgataa gatacattga
     1861 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     1921 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     1981 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aatgcatcca
     2041 cggggttggg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct
     2101 ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt
     2161 ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc
     2221 ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa
     2281 cggaagccgc acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag
     2341 cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca
     2401 gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc
     2461 tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc
     2521 tcgttgaccg aatcaccgac ctctctcccc aggatccgta cgatatcgcc atgaatccca
     2581 gtatgaagca gaaacaagaa gaaatcaaag agaatataaa gaatagttct gtcccaagaa
     2641 gaactctgaa gatgattcag ccttctgcat ctggatctct tgttggaaga gaaaatgagc
     2701 tgtccgcagg cttgtccaaa aggaaacatc ggaatgacca cttaacatct acaacttcca
     2761 gccctggggt tattgtccca gaatctagtg aaaataaaaa tcttggagga gtcacccagg
     2821 agtcatttga tcttatgatt aaagaaaatc catcctctca gtattggaag gaagtggcag
     2881 aaaaacggag aaaggcgctg ggcgggggat ccatggtgag caagggcgag gaggataaca
     2941 tggcctctct cccagcgaca catgagttac acatctttgg ctccatcaac ggtgtggact
     3001 ttgacatggt gggtcagggc accggcaatc caaatgatgg ttatgaggag ttaaacctga
     3061 agtccaccaa gggtgacctc cagttctccc cctggattct ggtccctcat atcgggtatg
     3121 gcttccatca gtacctgccc taccctgacg ggatgtcgcc tttccaggcc gctatggtag
     3181 atggctccgg ataccaagtc catcgcacaa tgcagtttga agatggtgcc tcccttactg
     3241 ttaactaccg ctacacctac gagggaagcc acatcaaagg agaggcccag gtgaagggga
     3301 ctggtttccc tgctgacggt cctgtgatga ccaactcgct gaccgctgcg gactggtgca
     3361 ggtcgaagaa gacttacccc aacgacaaaa ccatcatcag tacctttaag tggagttaca
     3421 ccactggaaa tggcaagcgc taccggagca ctgcgcggac cacctacacc tttgccaagc
     3481 caatggcggc taactatctg aagaaccagc cgatgtacgt gttccgtaag acggagctca
     3541 agcactccaa gaccgagctc aacttcaagg agtggcaaaa ggcctttacc gatgtgatgg
     3601 gcatggacga gctgtacaag ggatatccat cacactggcg gccgctcgag gccagcccgc
     3661 cgtcccccgc gcgatggagc ctgggacgga agcgcagagc cgacggcagg cgctggaggc
     3721 ccgaagacgc cgaggaggca gagcaccgcg gcgccgagcg cagacccgag agctttacca
     3781 ctcctgaagg ccctaaaccc cgttccagat gctctgactg ggcaagtgca gttgaagaag
     3841 atgaaatgag gaccagagtt aacaaagaaa tggcaagata taaaaggaaa ctcctcatca
     3901 atgactttgg aagagagaga aaaggcgggg gatccgacta caaggaccac gacggcgatt
     3961 ataaggatca cgacatcgac tacaaagacg acgatgacaa gggcgccagc agcgcctggt
     4021 cccaccctca gtttgagaag ggcggaggct ctggcggcgg aagcggagga tctgcttgga
     4081 gccaccccca gttcgaaaag tgataagttt aaaccctgca ggctgtgcct tctagttgcc
     4141 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     4201 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     4261 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     4321 atgctgggga tgcggtgggc tctatgggtc gacagtacta agctttacta gggacaggat
     4381 tggtgacaga aaagccccat ccttaggcct cctccttcct agtctcctga tattgggtct
     4441 aacccccacc tcctgttagg cagattcctt atctggtgac acacccccat ttcctggagc
     4501 catctctctc cttgccagaa cctctaaggt ttgcttacga tggagccaga gaggatcctg
     4561 ggagggagag cttggcaggg ggtgggaggg aaggggggga tgcgtgacct gcccggttct
     4621 cagtggccac cctgcgctac cctctcccag aacctgagct gctctgacgc ggctgtctgg
     4681 tgcgtttcac tgatcctggt gctgcagctt ccttacactt cccaagagga gaagcagttt
     4741 ggaaaaacaa aatcagaata agttggtcct gagttctaac tttggctctt cacctttcta
     4801 gtccccaatt tatattgttc ctccgtgcgt cagttttacc tgtgagataa ggccagtagc
     4861 cagccccgtc ctggcagggc tgtggtgagg aggggggtgt ccgtgtggaa aactcccttt
     4921 gtgagaatgg tgcgtcctag gtgttcacca ggtcgtggcc gcctctactc cctttctctt
     4981 tctccatcct tctttcctta aagagtcccc agtgctatct gggacatatt cctccgccca
     5041 gagcagggtc ccgcttccct aaggccctgc tctgggcttc tgggtttgag tccttggcaa
     5101 gcccaggaga ggcgctcagg cttccctgtc ccccttcctc gtccaccatc tcatgcccct
     5161 ggctctcctg ccccttccct acaggggttc ctggctctgc tctaagggcg aattctgcat
     5221 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5281 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5341 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5401 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5461 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5521 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5581 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5641 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5701 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5761 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5821 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5881 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5941 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6001 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6061 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6121 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6181 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6241 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6301 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6361 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6421 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6481 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6541 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6601 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6661 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6721 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6781 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6841 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6901 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6961 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7021 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7081 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7141 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7201 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     7261 ccctttcgtc
//
