LOCUS       sequence-378103-        7093 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor for targeted integration of 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 7093)
  TITLE     S-phase specific probe
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7093)
  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            "Unpublished"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)
FEATURES             Location/Qualifiers
     source          1..7093
                     /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"
     polyA_signal    3946..4170
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    4190..5026
                     /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(5103..5120)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5274..5862)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5354..5373)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6033..6824,6825..6893))
                     /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     6656..6675
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6894..6998)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7066..7084
                     /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 aaatgataag tttaaaccct gcaggctgtg ccttctagtt
     3961 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     4021 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     4081 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     4141 ggcatgctgg ggatgcggtg ggctctatgg gtcgacagta ctaagcttta ctagggacag
     4201 gattggtgac agaaaagccc catccttagg cctcctcctt cctagtctcc tgatattggg
     4261 tctaaccccc acctcctgtt aggcagattc cttatctggt gacacacccc catttcctgg
     4321 agccatctct ctccttgcca gaacctctaa ggtttgctta cgatggagcc agagaggatc
     4381 ctgggaggga gagcttggca gggggtggga gggaaggggg ggatgcgtga cctgcccggt
     4441 tctcagtggc caccctgcgc taccctctcc cagaacctga gctgctctga cgcggctgtc
     4501 tggtgcgttt cactgatcct ggtgctgcag cttccttaca cttcccaaga ggagaagcag
     4561 tttggaaaaa caaaatcaga ataagttggt cctgagttct aactttggct cttcaccttt
     4621 ctagtcccca atttatattg ttcctccgtg cgtcagtttt acctgtgaga taaggccagt
     4681 agccagcccc gtcctggcag ggctgtggtg aggagggggg tgtccgtgtg gaaaactccc
     4741 tttgtgagaa tggtgcgtcc taggtgttca ccaggtcgtg gccgcctcta ctccctttct
     4801 ctttctccat ccttctttcc ttaaagagtc cccagtgcta tctgggacat attcctccgc
     4861 ccagagcagg gtcccgcttc cctaaggccc tgctctgggc ttctgggttt gagtccttgg
     4921 caagcccagg agaggcgctc aggcttccct gtcccccttc ctcgtccacc atctcatgcc
     4981 cctggctctc ctgccccttc cctacagggg ttcctggctc tgctctaagg gcgaattctg
     5041 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5101 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5161 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5221 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5281 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5341 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5401 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5461 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5521 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5581 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5641 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5701 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5761 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5821 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5881 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5941 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6001 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6061 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6121 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6181 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6241 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6301 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6361 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6421 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6481 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6541 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6601 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6661 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6721 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6781 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6841 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6901 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6961 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7021 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     7081 aggccctttc gtc
//
