LOCUS       sequence-397579-        3422 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of eukaryotic genes of interest with Ef1a promoter 
            with Kpn1 as a PlayBack Restriction Enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3422)
  AUTHORS   Foran G, Hallam RD, Megaly M, Turgambayeva A, Necakov A
  TITLE     PlayBack cloning: simple, reversible, cost-effective cloning for the
            combinatorial assembly of complex expression constructs.
  JOURNAL   Biotechniques. 2023 Oct;75(4):168-178. doi: 10.2144/btn-2023-0042. 
            Epub 2023 Oct 10.
  PUBMED    37815818
REFERENCE   2  (bases 1 to 3422)
  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"; doi: 
            "10.2144/btn-2023-0042"; journalName: "Biotechniques"; date: 
            "2023-10"; volume: "75"; issue: "4"; pages: "168-178"
COMMENT     Sequence Label: PlayBack-Ef1aKpn1
FEATURES             Location/Qualifiers
     source          1..3422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    778..1002
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(1056..2234)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1065..2003
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(1088..1108)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        2287..2391
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2392..2460,2461..3252)
                     /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(2610..2629)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtat ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagccat ggtcgaccag gtaccgacca
      781 tagagcccac cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt
      841 cctgccccac cccacccccc agaatagaat gacacctact cagacaatgc gatgcaattt
      901 cctcatttta ttaggaaagg acagtgggag tggcaccttc cagggtcaag gaaggcacgg
      961 gggaggggca aacaacagat ggctggcaac tagaaggcac agtcattcat tcacaagctt
     1021 agtcaatcaa tctagactga gatccctagc attcctcacg acacctgaaa tggaagaaaa
     1081 aaactttgaa ccactgtctg aggcttgaga atgaaccaag atccaaactc aaaaagggca
     1141 aattccaagg agaattacat caagtgccaa gctggcctaa cttcagtctc cacccactca
     1201 gtgtggggaa actccatcgc ataaaacccc tccccccaac ctaaagacga cgtactccaa
     1261 aagctccaga actaatcgag gtgcctggac ggcgcccggt actccgtgga gtcacatgaa
     1321 gcgacggctg aggacggaaa ggcccttttc ctttgtgtgg gtgactcacc cgcccgctct
     1381 cccgagcgcc gcgtcctcca ttttgagctc cctgcagcag ggccgggaag cggccatctt
     1441 tccgctcacg caactggtgc cgaccgggcc agccttgccg cccagggcgg ggcgatacac
     1501 ggcggcgcga ggccaggcac cagagcaggc cggccagctt gagactaccc ccgtccgatt
     1561 ctcggtggcc gcgctcgcag gccccgcctc gccgaacatg tgcgctggga cgcacgggcc
     1621 ccgtcgccgc ccgcggcccc aaaaaccgaa ataccagtgt gcagatcttg gcccgcattt
     1681 acaagactat cttgccagaa aaaaagcgtc gcagcaggtc atcaaaaatt ttaaatggct
     1741 agagacttat cgaaagcagc gagacaggcg cgaaggtgcc accagattcg cacgcggcgg
     1801 ccccagcgcc caggccaggc ctcaactcaa gcacgaggcg aaggggctcc ttaagcgcaa
     1861 ggcctcgaac tctcccaccc acttccaacc cgaagctcgg gatcaagaat cacgtactgc
     1921 agccaggtgg aagtaattca aggcacgcaa gggccataac ccgtaaagag gccaggcccg
     1981 cgggaaccac acacggcact tacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg
     2041 ttcacggcga ctactgcact tatatacggt tctcccccac cctcgggaaa aaggcggagc
     2101 cagtacacga catcactttc ccagtttacc ccgcgccacc ttctctaggc accggttcaa
     2161 ttgccgaccc ctccccccaa cttctcgggg actgtgggcg atgtgcgctc tgcccactga
     2221 cgggcaccgg agccaattcc cggtaccata atatatctcg agttacgcgt aattcgggga
     2281 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2341 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     2401 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     2461 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     2521 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     2581 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     2641 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     2701 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2761 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     2821 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2881 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     2941 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3001 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3061 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3121 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3181 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3241 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     3301 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     3361 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3421 tc
//
