LOCUS       sequence-465496-        4044 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IVT template for making bicistronic modRNA encoding Flippase and 
            Zeocin resistance gene .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4044)
  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 4044)
  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: pIVT-Flp-2A-BleoR
FEATURES             Location/Qualifiers
     source          1..4044
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        22..126
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(127..195,196..987)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGYRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(345..364)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1158..1746
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1647..1666
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     regulatory      1818..1827
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1824..1829,1830..1850,1851..3119)
                     /codon_start=1
                     /product="nuclear-targeted site-specific recombinase"
                     /label=FLPo
                     /note="FLPo is a mouse codon-optimized fusion between the 
                     SV40 nuclear localization signal and a thermostable version
                     of the Saccharomyces cerevisiae site-specific recombinase 
                     FLP.  Recombination occurs at FRT sequences."
                     /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     CDS             3129..3182
                     /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             3183..3554
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="AKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDDV
                     TLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGRE
                     FALRDPAGNCVHFVAEEQD"
     polyA_signal    join(3671..4044,1..21)
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
ORIGIN
        1 gttttgctat gctgtatttt acgcggaacc cctatttgtt tatttttcta aatacattca
       61 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
      121 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
      181 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
      241 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
      301 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
      361 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
      421 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
      481 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
      541 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
      601 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
      661 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
      721 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
      781 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
      841 gggtatcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
      901 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
      961 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     1021 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     1081 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     1141 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     1201 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1261 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     1321 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1381 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     1441 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     1501 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     1561 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     1621 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     1681 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     1741 tggaaactcc tgggcaacgt gctggtctgt gtgctggccc atcactttgg caaagcacgc
     1801 taccggtcac gcgtgccgcc accatggctc ctaagaagaa gaggaaggtg atgagccagt
     1861 tcgacatcct gtgcaagacc ccccccaagg tgctggtgcg gcagttcgtg gagagattcg
     1921 agaggcccag cggcgagaag atcgccagct gtgccgccga gctgacctac ctgtgctgga
     1981 tgatcaccca caacggcacc gccatcaaga gggccacctt catgagctac aacaccatca
     2041 tcagcaacag cctgagcttc gacatcgtga acaagagcct gcagttcaag tacaagaccc
     2101 agaaggccac catcctggag gccagcctga agaagctgat ccccgcctgg gagttcacca
     2161 tcatccctta caacggccag aagcaccaga gcgacatcac cgacatcgtg tccagcctgc
     2221 agctgcagtt cgagagcagc gaggaggccg acaagggcaa cagccacagc aagaagatgc
     2281 tgaaggccct gctgtccgag ggcgagagca tctgggagat caccgagaag atcctgaaca
     2341 gcttcgagta caccagcagg ttcaccaaga ccaagaccct gtaccagttc ctgttcctgg
     2401 ccacattcat caactgcggc aggttcagcg acatcaagaa cgtggacccc aagagcttca
     2461 agctggtgca gaacaagtac ctgggcgtga tcattcagtg cctggtgacc gagaccaaga
     2521 caagcgtgtc caggcacatc tactttttca gcgccagagg caggatcgac cccctggtgt
     2581 acctggacga gttcctgagg aacagcgagc ccgtgctgaa gagagtgaac aggaccggca
     2641 acagcagcag caacaagcag gagtaccagc tgctgaagga caacctggtg cgcagctaca
     2701 acaaggccct gaagaagaac gccccctacc ccatcttcgc tatcaagaac ggccctaaga
     2761 gccacatcgg caggcacctg atgaccagct ttctgagcat gaagggcctg accgagctga
     2821 caaacgtggt gggcaactgg agcgacaaga gggcctccgc cgtggccagg accacctaca
     2881 cccaccagat caccgccatc cccgaccact acttcgccct ggtgtccagg tactacgcct
     2941 acgaccccat cagcaaggag atgatcgccc tgaaggacga gaccaacccc atcgaggagt
     3001 ggcagcacat cgagcagctg aagggcagcg ccgagggcag catcagatac cccgcctgga
     3061 acggcatcat cagccaggag gtgctggact acctgagcag ctacatcaac aggcggatcg
     3121 gaagcggaga aggtagaggt tctctcctca cttgtggtga tgttgaagaa aaccctggtc
     3181 cagccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg
     3241 agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg
     3301 tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca
     3361 acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg
     3421 tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc
     3481 cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg
     3541 aggagcagga ctaagcggcc gcactcctca ggtctcgagc tcaagcttcg aattcacccc
     3601 accagtgcag gctgcctatc agaaagtggt ggctggtgtg gctaatgccc tggcccacaa
     3661 gtatcactaa gctcgctttc ttgctgtcca atttctatta aaggttcctt tgttccctaa
     3721 gtccaactac taaactgggg gatattatga agggccttga gcatctggat tctgcctaat
     3781 aaaaaacatt tattttcatt gcaatgatgt atttaaatta tttctgaata ttttactaaa
     3841 aagggaatgt gggaggtcag tgcatttaaa acataaagaa atgaagagct agttcaaacc
     3901 ttgggaaaat acactatatc ttaaactcca tgaaagaagg tgaggctgca aacagctaat
     3961 gcacattggc aacagcccct gatgcctatg ccttattcat ccctcagaaa aggattcaag
     4021 tagaggcttt atttggaggt taaa
//
