LOCUS       sequence-364915-        7155 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor for both PiggyBac and Cp36 Recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7155)
  AUTHORS   Durrant MG, Fanton A, Tycko J, Hinks M, Chandrasekaran SS, Perry NT,
            Schaepe J, Du PP, Lotfy P, Bassik MC, Bintu L, Bhatt AS, Hsu PD
  TITLE     Systematic discovery of recombinases for efficient integration of 
            large DNA sequences into the human genome.
  JOURNAL   Nat Biotechnol. 2022 Oct 10. pii: 10.1038/s41587-022-01494-w. doi: 
            10.1038/s41587-022-01494-w.
  PUBMED    36217031
REFERENCE   2  (bases 1 to 7155)
  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: "Nat 
            Biotechnol. 2022 Oct 10. pii: 10.1038/s41587-022-01494-w. doi: 
            10.1038/s41587-022-01494-w."
COMMENT     Sequence Label: pJT371 PB ITR and Cp36 attD pEF Puro-2a-mCherr...
FEATURES             Location/Qualifiers
     source          1..7155
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     584..603
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(822..926)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(952..1407)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1089..1110)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1301..1320
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1534..1556
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1548..1565
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1549..1565
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   1695..1757
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2340..3518
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2571..3509
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3466..3486
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3526..4122
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3526..3545)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4022..4042
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4123..4179
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4186..4896
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4331..4349)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4599..4618
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4921..4938)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4927..5134
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5430..5464
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5955..5971)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5955..5971)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5968..5990)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5979..5995
                     /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)."
     promoter        complement(join(6003..6009,6010..6027,6028..6033))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6048..6069
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6186..6203)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6357..6945)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6437..6456)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7116..7155,1..752,753..821))
                     /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"
ORIGIN
        1 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
       61 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
      121 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
      181 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
      241 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
      301 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
      361 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
      421 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
      481 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
      541 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
      601 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
      661 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
      721 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
      781 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
      841 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
      901 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
      961 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     1021 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     1081 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     1141 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     1201 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     1261 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     1321 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     1381 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     1441 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     1501 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
     1561 ccagtgagcg cgcctcgttc attcacgttt ttgaacccgt ggaggacggg cagactcgcg
     1621 gtgcaaatgt gttttacagc gtgatggagc agatgaagat gctcgacacg ctgcagaaca
     1681 cgcagctaga ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg
     1741 tgtaaaattg acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa
     1801 tagatattaa gttttattat atttacactt acatactaat aataaattca acaaacaatt
     1861 tatttatgtt tatttattta ttaaaaaaaa caaaaactca aaatttcttc tataaagtaa
     1921 caaaactttt atgagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc
     1981 ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg
     2041 catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg
     2101 ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg
     2161 aaaaggcctc cacggccact agtttcgcac gattgcacct tgggagcctt aaccgctttt
     2221 gaatgtccgt cttagttaat ccctaattga acgctccaaa aggtgacttc caatagggat
     2281 ttattccttt taaaattaac ggcataatcg tgccgccgaa tgcaacctcg atcgagccgg
     2341 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     2401 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     2461 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     2521 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
     2581 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
     2641 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
     2701 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
     2761 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
     2821 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
     2881 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     2941 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     3001 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
     3061 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     3121 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     3181 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     3241 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     3301 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     3361 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     3421 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     3481 tggttcaaag tttttttctt ccatttcagg tgtcgtgaag ccaccatgac cgagtacaag
     3541 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     3601 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     3661 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     3721 gtcgcggacg acggcgccgc cgtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     3781 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     3841 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     3901 gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     3961 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     4021 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     4081 gaaggaccgc gcacctggtg catgacccgc aagcccggtg ccgccacgaa cttctctctg
     4141 ttaaagcaag caggtgacgt ggaagaaaac cccggtcccg gatccatggt gagcaagggc
     4201 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     4261 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     4321 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     4381 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     4441 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     4501 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     4561 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     4621 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     4681 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     4741 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     4801 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     4861 cactccaccg gcggcatgga cgagctgtac aagtaactcg agctagagct cgctgatcag
     4921 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     4981 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     5041 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     5101 aggattggga agacaatagc aggcatgctg gggacgacgt caggtggcac ttttcgatat
     5161 ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg aaataacaat
     5221 ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg cgtcattttg
     5281 actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca agcacgcctc
     5341 acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt
     5401 tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga ctatctttct
     5461 agggttaatc tagctgcatc aggatcatat cgtcgggtct tttttccggc tcagtcatcg
     5521 cccaagctgg cgctatctgg gcatcgggga ggaagaagcc cgtgcctttt cccgcgaggt
     5581 tgaagcggca tggaaagagt ttgccgagga tgactgctgc tgcattgacg ttgagcgaaa
     5641 acgcacgttt accatgatga ttcgggaagg tgtggccatg cacgccttta acggtgaact
     5701 gttcgttcag gccacctggg ataccagttc gtcgcggctt ttccggacac agttccggat
     5761 ggtcagcccg aagcgcatca gcaacccgaa caataccggc gacagccgga actgccgtgc
     5821 cggtgtgcag attaatgaca gcggtgcggc gctgggatat tacgtcagcg aggacgggta
     5881 tcctggctgg atgccgcaga aatggacatg gataccccgt gagttacccg gcgggcgcgc
     5941 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     6001 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     6061 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     6121 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     6181 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     6241 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     6301 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     6361 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     6421 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     6481 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     6541 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     6601 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     6661 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     6721 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6781 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6841 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6901 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6961 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     7021 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     7081 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     7141 cctatctcag cgatc
//
