LOCUS       sequence-362564-        4905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  phiC31-specific donor plasmid for the cloning of payloads.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4905)
  AUTHORS   Blanch-Asensio A, Grandela C, Brandao KO, de Korte T, Mei H, 
            Ariyurek Y, Yiangou L, Mol MPH, van Meer BJ, Kloet SL, Mummery CL, 
            Davis RP
  TITLE     STRAIGHT-IN enables high-throughput targeting of large DNA payloads 
            in human pluripotent stem cells.
  JOURNAL   Cell Rep Methods. 2022 Sep 22;2(10):100300. doi: 
            10.1016/j.crmeth.2022.100300. eCollection 2022 Oct 24.
  PUBMED    36313798
REFERENCE   2  (bases 1 to 4905)
  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: "Cell Rep 
            Methods."; date: "2022-09-22"; volume: "2(10):100300. doi"; pages: "
            10.1016/j.crmeth.2022.100300. eCollection 2022 Oct 24"
COMMENT     Sequence Label: pBR_attB(C31)_FRT
FEATURES             Location/Qualifiers
     source          1..4905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    102..123
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(138..143,144..161,162..168)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    176..192
                     /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)."
     primer_bind     181..203
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     200..216
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     200..216
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        248..458
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        270..466
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      317..452
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        553..1731
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          784..1722
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1679..1699
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1739..1748
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1750..1819
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    1826..1873
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    1958..1991
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     primer_bind     complement(1999..2015)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2029..2045)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2038..2060)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2258..2713
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2345..2364)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2555..2576
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2730..2749)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2849..2871
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2909..2927)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2995..3099
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3100..3168,3169..3960)
                     /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     complement(3318..3337)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4131..4719
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4620..4639
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4873..4890
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
       61 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
      121 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
      181 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcttaattcc
      241 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
      301 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
      361 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
      421 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tttgcaaaga
      481 tggataaagt tttaaacaga gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa
      541 ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
      601 gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta
      661 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
      721 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
      781 caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc
      841 gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt
      901 ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga
      961 gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc
     1021 tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg
     1081 ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg
     1141 gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg
     1201 cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct
     1261 gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc
     1321 cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc
     1381 tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa
     1441 agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc
     1501 aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg
     1561 ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg
     1621 cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc
     1681 aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg aggaattcgc
     1741 caccatggcc tcgaagccgc ggtgcgggtg ccagggcgtg cccttgggct ccccgggcgc
     1801 gtactccacc tcacccatcg gatccgaagt tcctattccg aagttcctat tctctagaaa
     1861 gtataggaac ttcatgatat tcggcaagca ggcatcgact agttaattaa gctagcgctg
     1921 caagaactct tcctcacgga tccgaagttc ctattccgaa gttcctattc ttcaaatagt
     1981 ataggaactt cagcgctcac tggccgtcgt tttacaaagg gcgaattcac tggccgtcgt
     2041 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     2101 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     2161 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     2221 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     2281 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     2341 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2401 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2461 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2521 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2581 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     2641 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     2701 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     2761 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     2821 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     2881 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     2941 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3001 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3061 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3121 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3181 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3241 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3301 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3361 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3421 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3481 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3541 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3601 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3661 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3721 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3781 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3841 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3901 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3961 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4021 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4081 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4141 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4201 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4261 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4321 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4381 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4441 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4501 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4561 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4621 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4681 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4741 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4801 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4861 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgc
//
