LOCUS       sequence-369405-        7763 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 (GGGGS)2 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 7763)
  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 7763)
  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-2xL
FEATURES             Location/Qualifiers
     source          1..7763
                     /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             4943..5278
                     /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(5427..5445)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(5966..5983)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5972..6196
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        6628..6732
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6733..6801,6802..7593)
                     /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(6951..6970)
                     /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 gggaggcggt ggatcggaac tgaccacact gtccggactg tctggagagc agggaccatc
     4981 cggcgacatg accacagagg aggattctgc cacacacatc aagttcagca agagggacga
     5041 ggacggaaga gagctggcag gagcaaccat ggagctgagg gatagctccg gcaagaccat
     5101 cagcacatgg atctccgacg gccacgtgaa ggatttctac ctgtatcccg gcaagtacac
     5161 ctttgtggag acagcagcac cagacggata tgaggtggca acccctatcg agtttacagt
     5221 gaacgaggac ggacaggtga ccgtggatgg agaggcaaca gagggcgatg cacacaccgg
     5281 atctagcgga tctatggagg aggacaacat ggccatcatc aaggagttca tgagattcaa
     5341 ggtgcacatg gagggcagcg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     5401 ccacccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc
     5461 cttcgcctgg gacatcctga gcccccagtt catgtacggc agcaaggcct acgtgaagca
     5521 ccccgccgac atccccgact acctgaagct gagcttcccc gagggcttca cctgggagag
     5581 agtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggacagca gcctgcagga
     5641 cggccagttc atctacaagg tgaagctgct gggcatcaac ttccccagcg acggccccgt
     5701 gatgcagaag aagaccatgg gctgggaggc cagcaccgag agaatgtacc ccgaggacgg
     5761 cgccctgaag ggcgagatca accagagact gaagctgaag gacggcggcc actacgacgc
     5821 cgaggtgaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
     5881 ggacatcaag ctggacatca ccagccacaa cgaggactag gtcaggccaa gcttggggat
     5941 caattctcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     6001 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     6061 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     6121 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     6181 gcggtgggct ctatggcttc tgaggcggaa agaaccagct gggcctacta gatagcggac
     6241 cccttaccga aacatcgccg cattctgcag aggagtcgaa cttagtgtat gtaaacttct
     6301 gacccactgg aattgtgata cagtgaatta taagtgaaat aatctgtctg taaacaattg
     6361 ttggaaaaat gacttgtgtc atgcacaaag tagatgtcct aactgacttg ccaaaactat
     6421 tgtttgttaa caagaaattt gtggagtagt tgaaaaacga gttttaatga ctccaactta
     6481 agtgtatgta aacttccgac ttcaactgta tagggatcca attttaacaa aatattaacg
     6541 cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     6601 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     6661 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     6721 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     6781 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     6841 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     6901 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     6961 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     7021 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     7081 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     7141 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     7201 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     7261 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     7321 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     7381 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     7441 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     7501 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     7561 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     7621 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     7681 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     7741 gtagaaaaga tcaaaggatc ttc
//
