LOCUS       sequence-372903-        7778 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)3 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 7778)
  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 7778)
  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-3xL
FEATURES             Location/Qualifiers
     source          1..7778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     179..198
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     438..459
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        1136..2318
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     primer_bind     2266..2286
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2342..2351
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2609..2638
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2708..3043
                     /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(3192..3210)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(3731..3748)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3737..3961
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4393..4497
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4498..4566,4567..5358)
                     /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(4716..4735)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5529..6117
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6018..6037
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6271..6288
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6662..6681
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    6680..6735
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     6781..6800
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(6900..7499)
                     /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(6983..7003)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     7480..7499
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(7500..7556)
                     /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(7566..7778,1..498,499..501,502..504))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MLSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(7566..7778,1..498,499..501,502..504))
                     /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(7608..7629)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg gccatgatat agacgttgtg
       61 gctgttgtag ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat
      121 gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt
      181 cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat
      241 ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcggctga agcactgcac
      301 gccgtaggtc agggtggtca cgagggtggg ccagggcacg ggcagcttgc cggtggtgca
      361 gatgaacttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct
      421 gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa
      481 cagctcctcg cccttgctca acatggtggc ctcaggtgca gaggtttcta cagggaaaga
      541 gagaaaggct acagttagtt tgctgtgatg atgctaagtg ccgaggtcag atgtctatgg
      601 attgtacacg gaatcccaac gcaggaacca aaaaacactc acccaaaaaa atggcgctaa
      661 ggccgagaga aaggcgaagt tccgtctacg gctatatacc ttgaaacagg ggcggcagaa
      721 gtgacgcaac gcggttgcgc ggacggcttc ctgtgttttc attggtcgaa gcgttctgcg
      781 cgtgcgcaga gattccccag ggaaggcggg ccctcttccg aatgtccaat ccggacattc
      841 ttccggttgg acctccgcgg tcagcgctgt gcccggcgag agatcacgtg gggcgcggag
      901 gcggtgctgc tggggcacgg ccgtccagcc tcggcggcca tatttttgag gggctgttca
      961 tctcgttcac acgctctgtc cgccatgttt gtgagtggaa gcgccattac cccttcaagc
     1021 gactgaaggc tgcagggcct ctggtggccc gcatggggag accagacccg ccaggcccgc
     1081 ctttccgcac tcagtccggg cttactttat tttgtgagac agggtctcgc ctagaggctc
     1141 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     1201 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     1261 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     1321 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg
     1381 tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc
     1441 cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
     1501 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
     1561 ttgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
     1621 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
     1681 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     1741 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     1801 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
     1861 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     1921 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     1981 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     2041 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     2101 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     2161 agtttcccca tactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     2221 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     2281 tggttcaaag tttttttctt ccatttaagg tgtcgtgaaa actaccccaa gctggcctct
     2341 ggccaccatg gatcaagcta gatcagcatt ctctaacttg tttggtggag aaccattgtc
     2401 atgtacccgg gccagcctgg ctcggcaagt agatggcgat aacagtcatg tggagatgaa
     2461 acttgctgta gatgaagaag aaaatgctga caataacaca aaggccaatg tcacaaaacc
     2521 aaaaaggtgt agtggaagta tctgctatgg gactattgct gtgatcgtct ttttcttgat
     2581 tggatttatg attggctact tgggctatga acaaaaactc atctcagaag aggatctggg
     2641 ttccggtggc tccgcgggag gcggtggatc gggaggcggt ggatcgggag gcggtggatc
     2701 ggaactgacc acactgtccg gactgtctgg agagcaggga ccatccggcg acatgaccac
     2761 agaggaggat tctgccacac acatcaagtt cagcaagagg gacgaggacg gaagagagct
     2821 ggcaggagca accatggagc tgagggatag ctccggcaag accatcagca catggatctc
     2881 cgacggccac gtgaaggatt tctacctgta tcccggcaag tacacctttg tggagacagc
     2941 agcaccagac ggatatgagg tggcaacccc tatcgagttt acagtgaacg aggacggaca
     3001 ggtgaccgtg gatggagagg caacagaggg cgatgcacac accggatcta gcggatctat
     3061 ggaggaggac aacatggcca tcatcaagga gttcatgaga ttcaaggtgc acatggaggg
     3121 cagcgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccacc cctacgaggg
     3181 cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat
     3241 cctgagcccc cagttcatgt acggcagcaa ggcctacgtg aagcaccccg ccgacatccc
     3301 cgactacctg aagctgagct tccccgaggg cttcacctgg gagagagtga tgaacttcga
     3361 ggacggcggc gtggtgaccg tgacccagga cagcagcctg caggacggcc agttcatcta
     3421 caaggtgaag ctgctgggca tcaacttccc cagcgacggc cccgtgatgc agaagaagac
     3481 catgggctgg gaggccagca ccgagagaat gtaccccgag gacggcgccc tgaagggcga
     3541 gatcaaccag agactgaagc tgaaggacgg cggccactac gacgccgagg tgaagaccac
     3601 ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtggaca tcaagctgga
     3661 catcaccagc cacaacgagg actaggtcag gccaagcttg gggatcaatt ctctagagct
     3721 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3781 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3841 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3901 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3961 gcttctgagg cggaaagaac cagctgggcc tactagatag cggacccctt accgaaacat
     4021 cgccgcattc tgcagaggag tcgaacttag tgtatgtaaa cttctgaccc actggaattg
     4081 tgatacagtg aattataagt gaaataatct gtctgtaaac aattgttgga aaaatgactt
     4141 gtgtcatgca caaagtagat gtcctaactg acttgccaaa actattgttt gttaacaaga
     4201 aatttgtgga gtagttgaaa aacgagtttt aatgactcca acttaagtgt atgtaaactt
     4261 ccgacttcaa ctgtataggg atccaatttt aacaaaatat taacgcttac aatttcctga
     4321 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatcagg tggcactttt
     4381 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4441 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4501 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4561 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4621 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4681 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4741 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4801 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4861 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4921 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4981 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5041 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5101 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5161 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5221 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5281 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5341 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5401 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5461 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5521 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5581 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5641 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5701 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5761 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5821 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5881 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5941 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6001 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6061 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6121 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6181 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     6241 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggatccc
     6301 tatacagttg aagtcggaag tttacataca ccttagccaa atacatttaa actcactttt
     6361 tcacaattcc tgacatttaa tcctagtaaa aattccctgt cttaggtcag ttaggatcac
     6421 cactttattt taagaatgtg aaatatcaga ataatagtag agagaatgat tcatttcagc
     6481 ttttatttct ttcatcacat tcccagtggg tcagaagttt acatacactc gactcctctg
     6541 cagaatgcgg cgatgtttcg gtaaggggtc ccgagtagct agttcatggc agccagcata
     6601 tggcatatgt tgccaaactc taaaccaaat actcattctg atgttttaaa tgatttgccc
     6661 tcccatatgt ccttccgagt gagagacaca aaaaattcca acacactatt gcaatgaaaa
     6721 taaatttcct ttattagcca gaagtcagat gctcaagggg cttcatgatg tccccataat
     6781 ttttggcaga gggaaaaaga tctcagtggt atttgtgagc cagggcattg gccacaccag
     6841 ccaccacctt ctgataggca gcctgcacct gaggagtgaa ttcacgacag gccttcgaat
     6901 caggcaccgg gcttgcgggt catgcaccag gtgcgcggtc cttcgggcac ctcgacgtcg
     6961 gcggtgacgg tgaagccgag ccgctcgtag aaggggaggt tgcggggcgc ggaggtctcc
     7021 aggaaggcgg gcaccccggc gcgctcggcc gcctccactc cggggagcac gacggcgctg
     7081 cccagaccct tgccctggtg gtcgggcgag acgccgacgg tggccaggaa ccacgcgggc
     7141 tccttgggcc ggtgcggcgc caggaggcct tccatctgtt gctgcgcggc cagccgggaa
     7201 ccgctcaact cggccatgcg cgggccgatc tcggcgaaca ccgcccccgc ttcgacgctc
     7261 tccggcgtgg tccagaccgc caccgcggcg ccgtcgtccg cgacccacac cttgccgatg
     7321 tcgagcccga cgcgcgtgag gaagagttct tgcagctcgg tgacccgctc gatgtggcgg
     7381 tccggatcga cggtgtggcg cgtggcgggg tagtcggcga acgcggcggc gagggtgcgt
     7441 acggccctgg ggacgtcgtc gcgggtggcg aggcgcaccg tgggcttgta ctcggtcatg
     7501 gggccaggat tctcctcgac gtcaccagcc tgcttcagca ggctgaagtt agtagctcca
     7561 ctgcccttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc
     7621 aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg
     7681 tagtggttgt cgggcagcag cacggggccg tcgccgattg gggtgttctg ctggtagtgg
     7741 tcggcgagct gcacgctgcc gtcctcgatg ttgtggcg
//
