LOCUS       sequence-462084-        8430 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CLYBL targeting construct with the Bxb1-GA landing pad cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8430)
  AUTHORS   Blanch-Asensio A, Ploessl DS, Wang NB, Mummery CL, Galloway KE, 
            Davis RP
  TITLE     STRAIGHT-IN Dual: a platform for dual, single-copy integrations of 
            DNA payloads and gene circuits into human induced pluripotent stem 
            cells.
  JOURNAL   bioRxiv [Preprint]. 2024 Oct 17:2024.10.17.616637. doi: 
            10.1101/2024.10.17.616637.
  PUBMED    39464154
REFERENCE   2  (bases 1 to 8430)
  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: "bioRxiv 
            [Preprint]. 2024 Oct 17:2024.10.17.616637. doi: 
            10.1101/2024.10.17.616637."
COMMENT     Sequence Label: CLYBL_Bxb1-GA_LP_Dual_TC
FEATURES             Location/Qualifiers
     source          1..8430
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    51..75
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     primer_bind     995..1012
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     996..1012
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1022..1069
                     /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)."
     promoter        1173..1672
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1208..1231)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1605..1624
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1679..1688
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1685..2380
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1818..1836)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2086..2105
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2384..2505
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2532..2579
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             2594..3190
                     /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     3087..3107
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3320..3441
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3457..3490
                     /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)"
     misc_feature    4350..4374
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      4462..5172
                     /label=oriV
                     /note="incP origin of replication"
     promoter        5193..5297
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5298..6092
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      6263..6851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6752..6771
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             7187..8335
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
ORIGIN
        1 agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg tggcaggata
       61 tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg gacgttttta
      121 atgtactggg gttgaacact ctgtgccagc aggagaatca tgccaatggg ccaatataca
      181 ttctgaccca cagtttcata ataaaataaa atggttgtgg ttgtaagcca ctatgtttca
      241 gagtggtttg ttacacagca ataaataact aatatagtag gcataccatc aagtccaaag
      301 taggtagaga agaatgtaaa tagcagagca aaacagcatg actggtggct gggaggctta
      361 aaactgggac aggatcagag tcatgaaaga agtcaaagaa atggttcaga agtaaggctg
      421 agactgactt acaaaagctg aaagtccctt taagttggtg tttggtgcat tggcaggggc
      481 aggtatggtg acttaaaaga gccatgctca acaagatcaa gcacaacaca atcacgggtc
      541 accccagcag accttagcga gtctagccat ttctttggtg gtggtcacag tcatgcttca
      601 gcccagtttc cacttggaca aatggtacat attttcaatg agatgaaaat taagatacaa
      661 tccatgtgct cagagagtga tcacagctct gactaaacac tgtgccccaa agtgttgagg
      721 aattgggaaa acctagctga gttagtggtc tcttttctgt tacaataaag ctcataatga
      781 aaattagcct tctttgttct tccccaagtc tttctttcta gacgaaacta ctttcaactg
      841 ttttaacttc cttactgtta acttccatat ttctagatag tatggtcaga ctgttatttc
      901 ttgactttta aatgtaagat attatctact gacttccttc tatgtaagat gaggattgag
      961 ctctcttacc cttctcccaa ctaagtcacg acgttgtaaa acgacggcca gtcttaatta
     1021 agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttca gcttgatcta
     1081 ctgagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     1141 cgagaagttg gggggagggg tcggcttcta ccgggtaggg gaggcgcttt tcccaaggca
     1201 gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg
     1261 gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc
     1321 cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc
     1381 gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag
     1441 caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc
     1501 cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg
     1561 gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca
     1621 aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgacaattgc
     1681 caccatggtg agcaagggcg aggcagtgat caaggagttc atgcggttca aggtgcacat
     1741 ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     1801 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttctcctg
     1861 ggacatcctg tcccctcagt tcatgtacgg ctccagggcc ttcaccaagc accccgccga
     1921 catccccgac tactataagc agtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     1981 cttcgaggac ggcggcgccg tgaccgtgac ccaggacacc tccctggagg acggcaccct
     2041 gatctacaag gtgaagctcc gcggcaccaa cttccctcct gacggccccg taatgcagaa
     2101 gaagacaatg ggctgggaag cgtccaccga gcggttgtac cccgaggacg gcgtgctgaa
     2161 gggcgacatt aagatggccc tgcgcctgaa ggacggcggc cgctacctgg cggacttcaa
     2221 gaccacctac aaggccaaga agcccgtgca gatgcccggc gcctacaacg tcgaccgcaa
     2281 gttggacatc acctcccaca acgaggacta caccgtggtg gaacagtacg aacgctccga
     2341 gggccgccac tccaccggcg gcatggacga gctgtacaag taaaacttgt ttattgcagc
     2401 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     2461 actgcattct agttgtggtt tgtccaaact catcaatgta tcttacatat gattcatgtc
     2521 tagatatacg tggtttgtct ggtcaaccac cgcggactca gtggtgtacg gtacaaaccc
     2581 atatgcgcgc cttaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     2641 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     2701 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     2761 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     2821 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     2881 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     2941 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa
     3001 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     3061 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     3121 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     3181 cggtgcctga cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     3241 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     3301 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     3361 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3421 caaactcatc aatgtatctt atgaagttcc tattccgaag ttcctattct tcaaatagta
     3481 taggaacttc gaacatccag ctgatatcca tcccctcctc ctcatccttc caacataaat
     3541 atattttggg attatatcaa cattcaatgt tacttaaagt gaccttgtaa atattttcac
     3601 aactgagcca tgtttgattt gtatacttat gtttacttta ctgtttttcc tgaagttaat
     3661 aattgccttg aatttattta tttctttaaa aatgtttcat tactcaggac tgtagtttac
     3721 attacgattc tttgtgttat acagttgatg ggtttctttt ctttcttaat ttctttaaaa
     3781 aatagagatg gggtcttact atattaccca ggctggtctt gaagtcctgg gctcaagtga
     3841 tcttcctgtc tcagcctacc aagtagctga gactataggt gcaaaaaagc cactatacct
     3901 ggctagttta caggttttaa caaatgcatt atgccacgta tccattatta caggatcaca
     3961 caagatattt tcattaccct gaaagcatcc ctgtgttcca ccaattcatc ctgcctccat
     4021 gagccgctgg caaccactga tctctatagt tttgcctttt ctaaaatgtc atataattgg
     4081 aatcatacag tctgtagcat tttcagacta gcttttaaaa tttggcaata tgcatttaag
     4141 gttcctcctt aaatgtgaag ggcatagcca atgtggcttg atagctcatt tctttttatt
     4201 ggtgaatatt tcattgtctg gatgttccac agtttgttta tccattcacc tattcaattt
     4261 gctttttttc tgtgtatcta tcactaattc aatactggac tctccaacag agccgggaca
     4321 cgaagtgatc cgtttaaact atcagtgttt gacaggatat attggcgggt aaacctaaga
     4381 gaaaagagcg tttattagaa taatcggata tttaaaaggg cgtgaaaagg tttatccgtt
     4441 cgtccatttg tatgtgccag ccgcctttgc gacgctcacc gggctggttg ccctcgccgc
     4501 tgggctggcg gccgtctatg gccctgcaaa cgcgccagaa acgccgtcga agccgtgtgc
     4561 gagacaccgc ggccgccggc gttgtggata cctcgcggaa aacttggccc tcactgacag
     4621 atgaggggcg gacgttgaca cttgaggggc cgactcaccc ggcgcggcgt tgacagatga
     4681 ggggcaggct cgatttcggc cggcgacgtg gagctggcca gcctcgcaaa tcggcgaaaa
     4741 cgcctgattt tacgcgagtt tcccacagat gatgtggaca agcctgggga taagtgccct
     4801 gcggtattga cacttgaggg gcgcgactac tgacagatga ggggcgcgat ccttgacact
     4861 tgaggggcag agtgctgaca gatgaggggc gcacctattg acatttgagg ggctgtccac
     4921 aggcagaaaa tccagcattt gcaagggttt ccgcccgttt ttcggccacc gctaacctgt
     4981 cttttaacct gcttttaaac caatatttat aaaccttgtt tttaaccagg gctgcgccct
     5041 gtgcgcgtga ccgcgcacgc cgaagggggg tgccccccct tctcgaaccc tcccggcccg
     5101 ctaacgcggg cctcccatcc ccccaggggc tgcgcccctc ggccgcgaac ggcctcaccc
     5161 caaaaatggc agcgctggcc aattcccgag tgcgcggaac ccctatttgt ttatttttct
     5221 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5281 attgaaaaag gaagagtatg gctaaaatga gaatatcacc ggaattgaaa aaactgatcg
     5341 aaaaataccg ctgcgtaaaa gatacggaag gaatgtctcc tgctaaggta tataagctgg
     5401 tgggagaaaa tgaaaaccta tatttaaaaa tgacggacag ccggtataaa gggaccacct
     5461 atgatgtgga acgggaaaag gacatgatgc tatggctgga aggaaagctg cctgttccaa
     5521 aggtcctgca ctttgaacgg catgatggct ggagcaatct gctcatgagt gaggccgatg
     5581 gcgtcctttg ctcggaagag tatgaagatg aacaaagccc tgaaaagatt atcgagctgt
     5641 atgcggagtg catcaggctc tttcactcca tcgacatatc ggattgtccc tatacgaata
     5701 gcttagacag ccgcttagcc gaattggatt acttactgaa taacgatctg gccgatgtgg
     5761 attgcgaaaa ctgggaagag gacactccat ttaaagatcc gcgcgagctg tatgattttt
     5821 taaagacgga aaagcccgaa gaggaacttg tcttttccca cggcgacctg ggagacagca
     5881 acatctttgt gaaagatggc aaagtaagtg gctttattga tcttgggaga agcggcaggg
     5941 cggacaagtg gtatgacatt gccttctgcg tccggtcgct cagggaggat atcggggaag
     6001 aacagtatgt cgagctattt tttgacttac tggggatcaa gcctgattgg gagaaaataa
     6061 aatattatat tttactggat gaattgtttt agctgtcaga ccaagtttac tcatatatac
     6121 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6181 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6241 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6301 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6361 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6421 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6481 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6541 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6601 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6661 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6721 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6781 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6841 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggcagat cctagatgtg
     6901 gcgcaacgat gccggcgaca agcaggagcg caccgacttc ttccgcatca agtgttttgg
     6961 ctctcaggcc gaggcccacg gcaagtattt gggcaagggg tcgctggtat tcgtgcaggg
     7021 caagattcgg aataccaagt acgagaagga cggccagacg gtctacggga ccgacttcat
     7081 tgccgataag gtggattatc tggacaccaa ggcaccaggc gggtcaaatc aggaataagg
     7141 gcacattgcc ccggcgtgag tcggggcaat cccgcaagga gggtgaatga atcggacgtt
     7201 tgaccggaag gcatacaggc aagaactgat cgacgcgggg ttttccgccg aggatgccga
     7261 aaccatcgca agccgcaccg tcatgcgtgc gccccgcgaa accttccagt ccgtcggctc
     7321 gatggtccag caagctacgg ccaagatcga gcgcgacagc gtgcaactgg ctccccctgc
     7381 cctgcccgcg ccatcggccg ccgtggagcg ttcgcgtcgt cttgaacagg aggcggcagg
     7441 tttggcgaag tcgatgacca tcgacacgcg aggaactatg acgaccaaga agcgaaaaac
     7501 cgccggcgag gacctggcaa aacaggtcag cgaggccaag caggccgcgt tgctgaaaca
     7561 cacgaagcag cagatcaagg aaatgcagct ttccttgttc gatattgcgc cgtggccgga
     7621 cacgatgcga gcgatgccaa acgacacggc ccgctctgcc ctgttcacca cgcgcaacaa
     7681 gaaaatcccg cgcgaggcgc tgcaaaacaa ggtcattttc cacgtcaaca aggacgtgaa
     7741 gatcacctac accggcgtcg agctgcgggc cgacgatgac gaactggtgt ggcagcaggt
     7801 gttggagtac gcgaagcgca cccctatcgg cgagccgatc accttcacgt tctacgagct
     7861 ttgccaggac ctgggctggt cgatcaatgg ccggtattac acgaaggccg aggaatgcct
     7921 gtcgcgccta caggcgacgg cgatgggctt cacgtccgac cgcgttgggc acctggaatc
     7981 ggtgtcgctg ctgcaccgct tccgcgtcct ggaccgtggc aagaaaacgt cccgttgcca
     8041 ggtcctgatc gacgaggaaa tcgtcgtgct gtttgctggc gaccactaca cgaaattcat
     8101 atgggagaag taccgcaagc tgtcgccgac ggcccgacgg atgttcgact atttcagctc
     8161 gcaccgggag ccgtacccgc tcaagctgga aaccttccgc ctcatgtgcg gatcggattc
     8221 cacccgcgtg aagaagtggc gcgagcaggt cggcgaagcc tgcgaagagt tgcgaggcag
     8281 cggcctggtg gaacacgcct gggtcaatga tgacctggtg cattgcaaac gctagggcct
     8341 tgtggggtca gttccggctg ggggttcagc agcccctgct cggatctgtt ggaccggaca
     8401 gtagtcatgg ttgatgggct gcctgtatcg
//
