LOCUS       sequence-425300-        7314 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An empty backbone for cloning of homology arms; ITR-flanked, 
            EF1alpha-driven expression of hygromycin-NLS-tdTomato.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7314)
  AUTHORS   Zhao Y, Pan Z, Hong Z, Sun M, Hong Y, Peng X, Li X, Wang X, Wang K
  TITLE     Protocol for scarless genome editing of human pluripotent stem cell 
            based on orthogonal selective reporters.
  JOURNAL   STAR Protoc. 2024 May 23;5(2):103084. doi: 
            10.1016/j.xpro.2024.103084.
  PUBMED    38787727
REFERENCE   2  (bases 1 to 7314)
  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: "STAR 
            Protoc."; date: "2024-05-23"; volume: "5(2):103084. doi"; pages: " 
            10.1016/j.xpro.2024.103084"
COMMENT     Sequence Label: P2A-mCherry-EhT backbone
FEATURES             Location/Qualifiers
     source          1..7314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(31..48)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    37..261
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   452..514
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     rep_origin      complement(593..1181)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(673..692)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1352..2143,2144..2212))
                     /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     1975..1994
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2213..2317)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2385..2403
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             2436..2492
                     /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             2493..3203
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     repeat_region   3208..3242
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    3519..3539
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        3540..4718
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3771..4709
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4666..4686
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4731..5753
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4731..5753
                     /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"
     CDS             5760..5816
                     /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             5826..5846
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5856..5876
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5883..7313
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
ORIGIN
        1 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
       61 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
      121 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
      181 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
      241 gggatgcggt gggctctatg ggcggccctg tccctcataa aagttttgtt actttataga
      301 agaaattttg agtttttgtt ttttttaata aataaataaa cataaataaa ttgtttgttg
      361 aatttattat tagtatgtaa gtgtaaatat aataaaactt aatatctatt caaattaata
      421 aataaacctc gatatacaga ccgataaaac acatgcgtca attttacaca tgattatctt
      481 taacgtacgt cacaatatga ttatctttct agggttaact ctctctgaat tccatgtgag
      541 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
      601 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
      661 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
      721 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
      781 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
      841 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
      901 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
      961 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     1021 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     1081 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     1141 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     1201 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     1261 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     1321 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     1381 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     1441 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     1501 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     1561 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     1621 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     1681 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     1741 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     1801 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     1861 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     1921 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     1981 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     2041 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     2101 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     2161 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     2221 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     2281 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     2341 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     2401 ggccctttcg tcggcgcggg taccggggat ccgtcgcgac gaacttctct ctgttaaagc
     2461 aagcaggaga tgttgaagaa aaccccgggc ctatggtgag caagggcgag gaggacaaca
     2521 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
     2581 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     2641 agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     2701 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     2761 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     2821 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     2881 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     2941 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     3001 tgaagctgaa ggacggcggc cactacgacg ccgaggtcaa gaccacctac aaggccaaga
     3061 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gctggacatc acctcccaca
     3121 acgaggacta caccatcgtg gaacagtacg agcgcgccga gggccgccac tccaccggcg
     3181 gcatggacga gctgtacaag taattaaccc tagaaagata gtctgcgtaa aattgacgca
     3241 tgcattcttg aaatattgct ctctctttct aaatagcgcg aatccgtcgc tgtgcattta
     3301 ggacatctca gtcgccgctt ggagctcccg tgaggcgtgc ttgtcaatgc ggtaagtgtc
     3361 actgattttg aactataacg accgcgtgag tcaaaatgac gcatgattat cttttacgtg
     3421 acttttaaga tttaactcat acgataatta tattgttatt tcatgttcta cttacgtgat
     3481 aacttattat atatatattt tcttgttata gatatcatca actttgtata gaaaagttgg
     3541 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     3601 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     3661 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     3721 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
     3781 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
     3841 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
     3901 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
     3961 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
     4021 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
     4081 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     4141 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     4201 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
     4261 gtctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     4321 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     4381 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     4441 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     4501 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     4561 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     4621 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     4681 tggttcaaag tttttttctt ccatttcagg tgtcgtgaaa gcttgccacc atgaaaaagc
     4741 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
     4801 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
     4861 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
     4921 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
     4981 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
     5041 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
     5101 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
     5161 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
     5221 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
     5281 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
     5341 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     5401 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     5461 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
     5521 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     5581 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     5641 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     5701 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaagctagcg
     5761 cgacgaattt tagtctactg aaacaagcgg gagacgtgga ggaaaaccct ggacctatcg
     5821 atggtccaaa aaagaagaga aaggtattcg atggtccaaa aaagaagaga aaggtattcg
     5881 atatggtgag caagggcgag gaggtcatca aagagttcat gcgcttcaag gtgcgcatgg
     5941 agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     6001 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg
     6061 acatcctgtc cccccagttc atgtacggct ccaaggcgta cgtgaagcac cccgccgaca
     6121 tccccgatta caagaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     6181 tcgaggacgg cggtctggtg accgtgaccc aggactcctc cctgcaggac ggcacgctga
     6241 tctacaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta atgcagaaga
     6301 agaccatggg ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg
     6361 gcgagatcca ccaggccctg aagctgaagg acggcggcca ctacctggtg gagttcaaga
     6421 ccatctacat ggccaagaag cccgtgcaac tgcccggcta ctactacgtg gacaccaagc
     6481 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgctccgagg
     6541 gccgccacca cctgttcctg gggcatggca ccggcagcac cggcagcggc agctccggca
     6601 ccgcctcctc cgaggacaac aacatggccg tcatcaaaga gttcatgcgc ttcaaggtgc
     6661 gcatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     6721 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg
     6781 cctgggacat cctgtccccc cagttcatgt acggctccaa ggcgtacgtg aagcaccccg
     6841 ccgacatccc cgattacaag aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     6901 tgaacttcga ggacggcggt ctggtgaccg tgacccagga ctcctccctg caggacggca
     6961 cgctgatcta caaggtgaag atgcgcggca ccaacttccc ccccgacggc cccgtaatgc
     7021 agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc
     7081 tgaagggcga gatccaccag gccctgaagc tgaaggacgg cggccgctac ctggtggagt
     7141 tcaagaccat ctacatggcc aagaagcccg tgcaactgcc cggctactac tacgtggaca
     7201 ccaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgct
     7261 ccgagggccg ccaccacctg ttcctgtacg gcatggacga gctgtacaag taat
//
