LOCUS       sequence-369404-        7748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Sleeping Beauty transposon system with constitutive bi-directional 
            promoter, one side expressing SpyCatcher003-sfCherry1-10 fused to 
            TfR through a single GGGGS linker and the other side expressing EGFP
            and puromycin resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7748)
  AUTHORS   Moghimianavval H, Patel C, Mohapatra S, Hwang SW, Kayikcioglu T, 
            Bashirzadeh Y, Liu AP, Ha T
  TITLE     Engineering Functional Membrane-Membrane Interfaces by InterSpy.
  JOURNAL   Small. 2022 May 26:e2202104. doi: 10.1002/smll.202202104.
  PUBMED    35618485
REFERENCE   2  (bases 1 to 7748)
  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: "Small. 2022
            May 26:e2202104. doi: 10.1002/smll.202202104."
COMMENT     Sequence Label: InterCatch-1xL
FEATURES             Location/Qualifiers
     source          1..7748
                     /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"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1134..1153
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    1152..1207
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     1253..1272
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(1372..1971)
                     /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(1455..1475)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1952..1971
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(1972..2028)
                     /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             complement(join(2038..2748,2749..2751,2752..2754))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MLSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2038..2748,2749..2751,2752..2754))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MLSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2080..2101)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2429..2448
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2688..2709
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        3386..4568
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     primer_bind     4516..4536
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4592..4601
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4859..4888
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4928..5263
                     /codon_start=1
                     /product="improved SpyCatcher variant that reacts with 
                     SpyTag003 at a rate approaching the diffusion limit (Keeble
                     et al., 2019)"
                     /label=SpyCatcher003
                     /translation="TTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMEL
                     RDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGE
                     ATEGDAHT"
     primer_bind     complement(5412..5430)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(5951..5968)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5957..6181
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        6613..6717
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6718..6786,6787..7578)
                     /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(6936..6955)
                     /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 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggatc cctatacagt
      781 tgaagtcgga agtttacata caccttagcc aaatacattt aaactcactt tttcacaatt
      841 cctgacattt aatcctagta aaaattccct gtcttaggtc agttaggatc accactttat
      901 tttaagaatg tgaaatatca gaataatagt agagagaatg attcatttca gcttttattt
      961 ctttcatcac attcccagtg ggtcagaagt ttacatacac tcgactcctc tgcagaatgc
     1021 ggcgatgttt cggtaagggg tcccgagtag ctagttcatg gcagccagca tatggcatat
     1081 gttgccaaac tctaaaccaa atactcattc tgatgtttta aatgatttgc cctcccatat
     1141 gtccttccga gtgagagaca caaaaaattc caacacacta ttgcaatgaa aataaatttc
     1201 ctttattagc cagaagtcag atgctcaagg ggcttcatga tgtccccata atttttggca
     1261 gagggaaaaa gatctcagtg gtatttgtga gccagggcat tggccacacc agccaccacc
     1321 ttctgatagg cagcctgcac ctgaggagtg aattcacgac aggccttcga atcaggcacc
     1381 gggcttgcgg gtcatgcacc aggtgcgcgg tccttcgggc acctcgacgt cggcggtgac
     1441 ggtgaagccg agccgctcgt agaaggggag gttgcggggc gcggaggtct ccaggaaggc
     1501 gggcaccccg gcgcgctcgg ccgcctccac tccggggagc acgacggcgc tgcccagacc
     1561 cttgccctgg tggtcgggcg agacgccgac ggtggccagg aaccacgcgg gctccttggg
     1621 ccggtgcggc gccaggaggc cttccatctg ttgctgcgcg gccagccggg aaccgctcaa
     1681 ctcggccatg cgcgggccga tctcggcgaa caccgccccc gcttcgacgc tctccggcgt
     1741 ggtccagacc gccaccgcgg cgccgtcgtc cgcgacccac accttgccga tgtcgagccc
     1801 gacgcgcgtg aggaagagtt cttgcagctc ggtgacccgc tcgatgtggc ggtccggatc
     1861 gacggtgtgg cgcgtggcgg ggtagtcggc gaacgcggcg gcgagggtgc gtacggccct
     1921 ggggacgtcg tcgcgggtgg cgaggcgcac cgtgggcttg tactcggtca tggggccagg
     1981 attctcctcg acgtcaccag cctgcttcag caggctgaag ttagtagctc cactgccctt
     2041 gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat
     2101 gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac tgggtgctca ggtagtggtt
     2161 gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag
     2221 ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga tgccgttctt
     2281 ctgcttgtcg gccatgatat agacgttgtg gctgttgtag ttgtactcca gcttgtgccc
     2341 caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt
     2401 gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat
     2461 ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat
     2521 gtggtcgggg tagcggctga agcactgcac gccgtaggtc agggtggtca cgagggtggg
     2581 ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt
     2641 ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag
     2701 ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca acatggtggc
     2761 ctcaggtgca gaggtttcta cagggaaaga gagaaaggct acagttagtt tgctgtgatg
     2821 atgctaagtg ccgaggtcag atgtctatgg attgtacacg gaatcccaac gcaggaacca
     2881 aaaaacactc acccaaaaaa atggcgctaa ggccgagaga aaggcgaagt tccgtctacg
     2941 gctatatacc ttgaaacagg ggcggcagaa gtgacgcaac gcggttgcgc ggacggcttc
     3001 ctgtgttttc attggtcgaa gcgttctgcg cgtgcgcaga gattccccag ggaaggcggg
     3061 ccctcttccg aatgtccaat ccggacattc ttccggttgg acctccgcgg tcagcgctgt
     3121 gcccggcgag agatcacgtg gggcgcggag gcggtgctgc tggggcacgg ccgtccagcc
     3181 tcggcggcca tatttttgag gggctgttca tctcgttcac acgctctgtc cgccatgttt
     3241 gtgagtggaa gcgccattac cccttcaagc gactgaaggc tgcagggcct ctggtggccc
     3301 gcatggggag accagacccg ccaggcccgc ctttccgcac tcagtccggg cttactttat
     3361 tttgtgagac agggtctcgc ctagaggctc cggtgcccgt cagtgggcag agcgcacatc
     3421 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     3481 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     3541 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     3601 tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg
     3661 ttatggccct tgcgtgcctt gaattacttc cacgcccctg gctgcagtac gtgattcttg
     3721 atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc
     3781 cttcgcctcg tgcttgagtt gaggcctggc ttgggcgctg gggccgccgc gtgcgaatct
     3841 ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt
     3901 gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc
     3961 tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc
     4021 gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt
     4081 ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct
     4141 gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg
     4201 gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt
     4261 cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg
     4321 agtaccgggc gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt
     4381 taggttgggg ggaggggttt tatgcgatgg agtttcccca tactgagtgg gtggagactg
     4441 aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg
     4501 gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt ccatttaagg
     4561 tgtcgtgaaa actaccccaa gctggcctct ggccaccatg gatcaagcta gatcagcatt
     4621 ctctaacttg tttggtggag aaccattgtc atgtacccgg gccagcctgg ctcggcaagt
     4681 agatggcgat aacagtcatg tggagatgaa acttgctgta gatgaagaag aaaatgctga
     4741 caataacaca aaggccaatg tcacaaaacc aaaaaggtgt agtggaagta tctgctatgg
     4801 gactattgct gtgatcgtct ttttcttgat tggatttatg attggctact tgggctatga
     4861 acaaaaactc atctcagaag aggatctggg ttccggtggc tccgcgggag gcggtggatc
     4921 ggaactgacc acactgtccg gactgtctgg agagcaggga ccatccggcg acatgaccac
     4981 agaggaggat tctgccacac acatcaagtt cagcaagagg gacgaggacg gaagagagct
     5041 ggcaggagca accatggagc tgagggatag ctccggcaag accatcagca catggatctc
     5101 cgacggccac gtgaaggatt tctacctgta tcccggcaag tacacctttg tggagacagc
     5161 agcaccagac ggatatgagg tggcaacccc tatcgagttt acagtgaacg aggacggaca
     5221 ggtgaccgtg gatggagagg caacagaggg cgatgcacac accggatcta gcggatctat
     5281 ggaggaggac aacatggcca tcatcaagga gttcatgaga ttcaaggtgc acatggaggg
     5341 cagcgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccacc cctacgaggg
     5401 cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat
     5461 cctgagcccc cagttcatgt acggcagcaa ggcctacgtg aagcaccccg ccgacatccc
     5521 cgactacctg aagctgagct tccccgaggg cttcacctgg gagagagtga tgaacttcga
     5581 ggacggcggc gtggtgaccg tgacccagga cagcagcctg caggacggcc agttcatcta
     5641 caaggtgaag ctgctgggca tcaacttccc cagcgacggc cccgtgatgc agaagaagac
     5701 catgggctgg gaggccagca ccgagagaat gtaccccgag gacggcgccc tgaagggcga
     5761 gatcaaccag agactgaagc tgaaggacgg cggccactac gacgccgagg tgaagaccac
     5821 ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtggaca tcaagctgga
     5881 catcaccagc cacaacgagg actaggtcag gccaagcttg gggatcaatt ctctagagct
     5941 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     6001 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     6061 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     6121 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     6181 gcttctgagg cggaaagaac cagctgggcc tactagatag cggacccctt accgaaacat
     6241 cgccgcattc tgcagaggag tcgaacttag tgtatgtaaa cttctgaccc actggaattg
     6301 tgatacagtg aattataagt gaaataatct gtctgtaaac aattgttgga aaaatgactt
     6361 gtgtcatgca caaagtagat gtcctaactg acttgccaaa actattgttt gttaacaaga
     6421 aatttgtgga gtagttgaaa aacgagtttt aatgactcca acttaagtgt atgtaaactt
     6481 ccgacttcaa ctgtataggg atccaatttt aacaaaatat taacgcttac aatttcctga
     6541 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatcagg tggcactttt
     6601 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     6661 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     6721 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     6781 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     6841 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     6901 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     6961 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     7021 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     7081 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     7141 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     7201 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     7261 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     7321 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     7381 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     7441 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     7501 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     7561 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     7621 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     7681 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     7741 ggatcttc
//
