LOCUS       sequence-364902-        7158 bp    DNA     circular SYN 14-APR-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 7158)
  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 7158)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pJT396 PB ITR and Cp36 attD pEF Puro-2a-tagBFP...
FEATURES             Location/Qualifiers
     source          1..7158
                     /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"
     primer_bind     complement(4924..4941)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4930..5137
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5433..5467
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5958..5974)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5958..5974)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5971..5993)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5982..5998
                     /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(6006..6012,6013..6030,6031..6036))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6051..6072
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6189..6206)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6360..6948)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6440..6459)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7119..7158,1..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 tagtaaggga
     4201 gaggagttga taaaagagaa tatgcacatg aaactgtata tggaagggac agtggacaac
     4261 catcacttca agtgcacctc tgagggagaa ggaaagccat acgaaggcac ccagactatg
     4321 cgaattaagg tcgtggaggg agggcctctc cccttcgcct tcgatattct tgcaacatct
     4381 ttcctctacg ggagcaaaac cttcatcaat cacacacaag gcatacctga cttttttaag
     4441 cagagttttc cagagggctt tacctgggag cgtgtgacta catacgaaga tgggggtgtg
     4501 ctgacggcta cacaggatac ttcacttcag gacggttgtc tgatctacaa cgtcaagatc
     4561 aggggggtca atttcacctc gaatggacca gtaatgcaaa aaaagaccct gggatgggaa
     4621 gcttttactg aaactctcta tcccgccgac ggcggcctcg aaggcagaaa cgatatggct
     4681 ttgaagctgg ttggcgggtc ccatctaatt gccaatgcca aaaccacata taggagcaaa
     4741 aagcctgcaa agaacctgaa gatgccgggt gtgtactatg ttgactatag attagagcgg
     4801 atcaaagagg caaataacga aacgtacgtg gagcagcacg aggtagccgt ggcgcgctac
     4861 tgcgatttac cctccaaact gggccataaa ctgaactaac tcgagctaga gctcgctgat
     4921 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     4981 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     5041 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     5101 gggaggattg ggaagacaat agcaggcatg ctggggacga cgtcaggtgg cacttttcga
     5161 tatctataac aagaaaatat atatataata agttatcacg taagtagaac atgaaataac
     5221 aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt
     5281 ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc
     5341 ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct
     5401 atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt
     5461 tctagggtta atctagctgc atcaggatca tatcgtcggg tcttttttcc ggctcagtca
     5521 tcgcccaagc tggcgctatc tgggcatcgg ggaggaagaa gcccgtgcct tttcccgcga
     5581 ggttgaagcg gcatggaaag agtttgccga ggatgactgc tgctgcattg acgttgagcg
     5641 aaaacgcacg tttaccatga tgattcggga aggtgtggcc atgcacgcct ttaacggtga
     5701 actgttcgtt caggccacct gggataccag ttcgtcgcgg cttttccgga cacagttccg
     5761 gatggtcagc ccgaagcgca tcagcaaccc gaacaatacc ggcgacagcc ggaactgccg
     5821 tgccggtgtg cagattaatg acagcggtgc ggcgctggga tattacgtca gcgaggacgg
     5881 gtatcctggc tggatgccgc agaaatggac atggataccc cgtgagttac ccggcgggcg
     5941 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     6001 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     6061 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6121 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     6181 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     6241 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     6301 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6361 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6421 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6481 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6541 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6601 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6661 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6721 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6781 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6841 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6901 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6961 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     7021 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     7081 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     7141 gcacctatct cagcgatc
//
