LOCUS       sequence-262549-        8366 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one Sleeping Beauty vector for stable transfection of 
            eukaryotic cells with hygromycin restistance. CMV protomor drives 
            SB100X transposase for transposon integration..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8366)
  AUTHORS   Flies AS, Darby JM, Lennard PR, Murphy PR, Ong CEB, Pinfold TL, De 
            Luca A, Lyons AB, Woods GM, Patchett AL
  TITLE     A novel system to map protein interactions reveals evolutionarily 
            conserved immune evasion pathways on transmissible cancers.
  JOURNAL   Sci Adv. 2020 Jul 1;6(27). pii: 6/27/eaba5031. doi: 
            10.1126/sciadv.aba5031. Print 2020 Jul.
  PUBMED    32937435
REFERENCE   2  (bases 1 to 8366)
  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: "Sci Adv."; 
            date: "2020-07-1"; volume: "6(27). pii: 6/27/eaba5031. doi"; pages: 
            " 10.1126/sciadv.aba5031. Print 2020 Jul"
COMMENT     Sequence Label: pAF123
FEATURES             Location/Qualifiers
     source          1..8366
                     /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..2397)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(1372..2397)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        3029..4211
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     primer_bind     4159..4179
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4237..4246
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4486..4503)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4492..4716
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        5078..5381
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5382..5585
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5536..5556
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5582..5606
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          5723..5819
                     /label=SV40 intron
                     /note="modified SV40 intron with splice donor and acceptor 
                     sites"
     primer_bind     5876..5895
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5876..5894
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5905..6927
                     /codon_start=1
                     /product="hyperactive variant of the Sleeping Beauty 
                     transposase (Mates et al., 2009)"
                     /label=SB100X
                     /translation="MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRK
                     YKHHGTTQPSYRSGRRRVLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVK
                     RVLYRHNLKGHSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHN
                     DHRYVWRKKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGTGALHKIDGIMDAVQYVDIL
                     KQHLKTSVRKLKLGRKWVFQHDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENL
                     WAELKKRVRARRPTNLTQLHQLCQEEWAKIHPNYCGKLVEGYPKRLTQVKQFKGNATKY
                     "
     polyA_signal    6944..7078
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6968..6987)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7022..7041
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        7231..7335
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7336..7404,7405..8196)
                     /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(7554..7573)
                     /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 actattcctt
     1381 tgccctcgga cgagtgctgg ggcgtcggtt tccactatcg gcgagtactt ctacacagcc
     1441 atcggtccag acggccgcgc ttctgcgggc gatttgtgta cgcccgacag tcccggctcc
     1501 ggatcggacg attgcgtcgc atcgaccctg cgcccaagct gcatcatcga aattgccgtc
     1561 aaccaagctc tgatagagtt ggtcaagacc aatgcggagc atatacgccc ggagccgcgg
     1621 cgatcctgca agctccggat gcctccgctc gaagtagcgc gtctgctgct ccatacaagc
     1681 caaccacggc ctccagaaga agatgttggc gacctcgtat tgggaatccc cgaacatcgc
     1741 ctcgctccag tcaatgaccg ctgttatgcg gccattgtcc gtcaggacat tgttggagcc
     1801 gaaatccgcg tgcacgaggt gccggacttc ggggcagtcc tcggcccaaa gcatcagctc
     1861 atcgagagcc tgcgcgacgg acgcactgac ggtgtcgtcc atcacagttt gccagtgata
     1921 cacatgggga tcagcaatcg cgcatatgaa atcacgccat gtagtgtatt gaccgattcc
     1981 ttgcggtccg aatgggccga acccgctcgt ctggctaaga tcggccgcag cgatcgcatc
     2041 catggcctcc gcgaccggct gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa
     2101 cgtgacaccc tgtgcacggc gggagatgca ataggtcagg ctctcgctga attccccaat
     2161 gtcaagcact tccggaatcg ggagcgcggc cgatgcaaag tgccgataaa cataacgatc
     2221 tttgtagaaa ccatcggcgc agctatttac ccgcaggaca tatccacgcc ctcctacatc
     2281 gaagctgaaa gcacgagatt cttcgccctc cgagagctgc atcaggtcgg agacgctgtc
     2341 gaacttttcg atcagaaact tctcgacaga cgtcgcggtg agttcaggct ttttcatggt
     2401 ggcctcaggt gcagaggttt ctacagggaa agagagaaag gctacagtta gtttgctgtg
     2461 atgatgctaa gtgccgaggt cagatgtcta tggattgtac acggaatccc aacgcaggaa
     2521 ccaaaaaaca ctcacccaaa aaaatggcgc taaggccgag agaaaggcga agttccgtct
     2581 acggctatat accttgaaac aggggcggca gaagtgacgc aacgcggttg cgcggacggc
     2641 ttcctgtgtt ttcattggtc gaagcgttct gcgcgtgcgc agagattccc cagggaaggc
     2701 gggccctctt ccgaatgtcc aatccggaca ttcttccggt tggacctccg cggtcagcgc
     2761 tgtgcccggc gagagatcac gtggggcgcg gaggcggtgc tgctggggca cggccgtcca
     2821 gcctcggcgg ccatattttt gaggggctgt tcatctcgtt cacacgctct gtccgccatg
     2881 tttgtgagtg gaagcgccat taccccttca agcgactgaa ggctgcaggg cctctggtgg
     2941 cccgcatggg gagaccagac ccgccaggcc cgcctttccg cactcagtcc gggcttactt
     3001 tattttgtga gacagggtct cgcctagagg ctccggtgcc cgtcagtggg cagagcgcac
     3061 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag
     3121 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     3181 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     3241 gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac
     3301 gggttatggc ccttgcgtgc cttgaattac ttccacgccc ctggctgcag tacgtgattc
     3361 ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag
     3421 ccccttcgcc tcgtgcttga gttgaggctt ggcctgggcg ctggggccgc cgcgtgcgaa
     3481 tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt
     3541 tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag
     3601 atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc
     3661 agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt
     3721 agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc
     3781 cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc
     3841 ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg
     3901 agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca
     3961 cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg agtacgtcgt
     4021 ctttaggttg gggggagggg ttttatgcga tggagtttcc ccatactgag tgggtggaga
     4081 ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt
     4141 ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt cttccattta
     4201 aggtgtcgtg aaaactaccc caagctggcc tctgaggcca ccatggctcg agtttcgaac
     4261 tcaattatac tgaattgttt catcataggg ttcaacattg tgataaatca ggacttcaat
     4321 gtctagtgtt cagtaaatac cttgtagggc tctccaaaaa ggggaatctt ttcaggaaat
     4381 gcctctttct cttggtgggc ttcctggttg cgtctttcct tctctctaat tcgaaggcct
     4441 gtcaggccaa gcttggggat caattctcta gagctcgctg atcagcctcg actgtgcctt
     4501 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     4561 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     4621 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     4681 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     4741 gggcctacta gatagcggac cccttaccga aacatcgccg cattctgcag aggagtcgaa
     4801 cttagtgtat gtaaacttct gacccactgg aattgtgata cagtgaatta taagtgaaat
     4861 aatctgtctg taaacaattg ttggaaaaat gacttgtgtc atgcacaaag tagatgtcct
     4921 aactgacttg ccaaaactat tgtttgttaa caagaaattt gtggagtagt tgaaaaacga
     4981 gttttaatga ctccaactta agtgtatgta aacttccgac ttcaactgta tagggatcca
     5041 attttaacaa aatattcgag cttgcatgcc tgcaggtcgt tacataactt acggtaaatg
     5101 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     5161 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     5221 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     5281 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     5341 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     5401 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     5461 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     5521 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     5581 gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc
     5641 atagaagaca ccgggaccga tccagcctcc ggactctaga ggatccggta ctcgaggaac
     5701 tgaaaaacca gaaagttaac tggtaagttt agtctttttg tcttttattt caggtcccgg
     5761 atccggtggt ggtgcaaatc aaagaactgc tcctcagtgg atgttgcctt tacttctagg
     5821 cctgtacgga agtgttactt ctgctctaaa agctgcggaa ttgtacccgc ggaattaata
     5881 cgactcacta tagggactag taccatggga aaatcaaaag aaatcagcca agacctcaga
     5941 aaaagaattg tagacctcca caagtctggt tcatccttgg gagcaatttc caaacgcctg
     6001 gcggtaccac gttcatctgt acaaacaata gtacgcaagt ataaacacca tgggaccacg
     6061 cagccgtcat accgctcagg aaggagacgc gttctgtctc ctagagatga acgtactttg
     6121 gtgcgaaaag tgcaaatcaa tcccagaaca acagcaaagg accttgtgaa gatgctggag
     6181 gaaacaggta caaaagtatc tatatccaca gtaaaacgag tcctatatcg acataacctg
     6241 aaaggccact cagcaaggaa gaagccactg ctccaaaacc gacataagaa agccagacta
     6301 cggtttgcaa ctgcacatgg ggacaaagat cgtacttttt ggagaaatgt cctctggtct
     6361 gatgaaacaa aaatagaact gtttggccat aatgaccatc gttatgtttg gaggaagaag
     6421 ggggaggctt gcaagccgaa gaacaccatc ccaaccgtga agcacggggg tggcagcatc
     6481 atgttgtggg ggtgctttgc tgcaggaggg actggtgcac ttcacaaaat agatggcatc
     6541 atggacgcgg tgcagtatgt ggatatattg aagcaacatc tcaagacatc agtcaggaag
     6601 ttaaagcttg gtcgcaaatg ggtcttccaa cacgacaatg accccaagca tacttccaaa
     6661 gttgtggcaa aatggcttaa ggacaacaaa gtcaaggtat tggagtggcc atcacaaagc
     6721 cctgacctca atcctataga aaatttgtgg gcagaactga aaaagcgtgt gcgagcaagg
     6781 aggcctacaa acctgactca gttacaccag ctctgtcagg aggaatgggc caaaattcac
     6841 ccaaattatt gtgggaagct tgtggaaggc tacccgaaac gtttgaccca agttaaacaa
     6901 tttaaaggca atgctaccaa atactagggg ccctaaccgc ggggatccag acatgataag
     6961 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     7021 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     7081 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     7141 acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     7201 gcatcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     7261 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     7321 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     7381 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     7441 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     7501 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     7561 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     7621 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     7681 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     7741 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     7801 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     7861 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     7921 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     7981 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     8041 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     8101 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     8161 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     8221 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     8281 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     8341 cccgtagaaa agatcaaagg atcttc
//
