LOCUS       sequence-389607-        7854 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-specific donor plasmid for integrating a transcription unit for
            constitutive expression of tdTomato reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7854)
  AUTHORS   Blanch-Asensio A, Grandela C, Mummery CL, Davis RP
  TITLE     STRAIGHT-IN: a platform for rapidly generating panels of genetically
            modified human pluripotent stem cell lines.
  JOURNAL   Nat Protoc. 2024 Aug 23. doi: 10.1038/s41596-024-01039-2.
  PUBMED    39179886
REFERENCE   2  (bases 1 to 7854)
  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: "Nat Protoc.
            2024 Aug 23. doi: 10.1038/s41596-024-01039-2."
COMMENT     Sequence Label: Bxb1_tdTomato
FEATURES             Location/Qualifiers
     source          1..7854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        253..463
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        275..471
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      322..457
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        558..1736
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          789..1727
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1684..1704
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1744..1753
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1756..1793
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    complement(1802..1835)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        1875..2178
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2719..2741
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     CDS             2796..4226
                     /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"
     primer_bind     complement(4247..4263)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4248..4264)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     protein_bind    complement(4275..4299)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    4330..4918
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4383..4403)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4801..4812)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(4943..4975)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(4983..5000)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4983..4999)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4992..5014)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5212..5667
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5299..5318)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5509..5530
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5684..5703)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5803..5825
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5863..5881)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5949..6053
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6054..6122,6123..6914)
                     /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(6272..6291)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7085..7673
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7574..7593
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7827..7844
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctta
      241 attccctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
      301 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
      361 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
      421 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctttgc
      481 aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct
      541 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
      601 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
      661 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
      721 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
      781 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc
      841 cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc
      901 gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg
      961 cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc
     1021 gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg
     1081 cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta
     1141 tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg
     1201 cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc
     1261 ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc
     1321 tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag
     1381 ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa
     1441 ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc
     1501 cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg
     1561 aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag
     1621 cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca
     1681 ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaggaa
     1741 ttcgccacca tggccggctt gtcgacgacg gcggtctccg tcgtcaggat catccggatc
     1801 cataacttcg tatagcatac attatacgaa gttatcatga tattcggcaa gcaggcatcg
     1861 actagttaat taagcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     1921 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     1981 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     2041 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     2101 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     2161 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
     2221 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
     2281 ggggcggggg gggggggggc gcgcgccrgg sggggcgggg cggggcgrgg ggcggggcgg
     2341 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
     2401 tatggcgagg cggcggcggc ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc
     2461 gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa
     2521 tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga
     2581 agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc
     2641 gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg
     2701 ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg
     2761 ctaaccatgt tcatgccttc ttctttttcc tacagatggt gagcaagggc gaggaggtca
     2821 tcaaagagtt catgcgcttc aaggtgcgca tggagggctc catgaacggc cacgagttcg
     2881 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
     2941 tgaccaaggg cggccccctg cccttcgcct gggacatcct gtccccccag ttcatgtacg
     3001 gctccaaggc gtacgtgaag caccccgccg acatccccga ttacaagaag ctgtccttcc
     3061 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggtctg gtgaccgtga
     3121 cccaggactc ctccctgcag gacggcacgc tgatctacaa ggtgaagatg cgcggcacca
     3181 acttcccccc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctccaccg
     3241 agcgcctgta cccccgcgac ggcgtgctga agggcgagat ccaccaggcc ctgaagctga
     3301 aggacggcgg ccactacctg gtggagttca agaccatcta catggccaag aagcccgtgc
     3361 aactgcccgg ctactactac gtggacacca agctggacat cacctcccac aacgaggact
     3421 acaccatcgt ggaacagtac gagcgctccg agggccgcca ccacctgttc ctggggcatg
     3481 gcaccggcag caccggcagc ggcagctccg gcaccgcctc ctccgaggac aacaacatgg
     3541 ccgtcatcaa agagttcatg cgcttcaagg tgcgcatgga gggctccatg aacggccacg
     3601 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     3661 tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc ccccagttca
     3721 tgtacggctc caaggcgtac gtgaagcacc ccgccgacat ccccgattac aagaagctgt
     3781 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggtctggtga
     3841 ccgtgaccca ggactcctcc ctgcaggacg gcacgctgat ctacaaggtg aagatgcgcg
     3901 gcaccaactt cccccccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct
     3961 ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg cgagatccac caggccctga
     4021 agctgaagga cggcggccgc tacctggtgg agttcaagac catctacatg gccaagaagc
     4081 ccgtgcaact gcccggctac tactacgtgg acaccaagct ggacatcacc tcccacaacg
     4141 aggactacac catcgtggaa cagtacgagc gctccgaggg ccgccaccac ctgttcctgt
     4201 acggcatgga cgagctgtac aagtaagaat tcgatatcaa gcttatcgat accgtcgacc
     4261 tcgagcctct agatacccag ctttcttgta caaagtggtt gatatccagc acagtggcgg
     4321 ccgctcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     4381 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     4441 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     4501 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     4561 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     4621 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     4681 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca
     4741 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     4801 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     4861 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggacgc ctccccgcct
     4921 gctgcaagaa ctcttcctca cgataacttc gtataggaga ctttatacga agttaagcgc
     4981 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     5041 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     5101 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     5161 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     5221 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5281 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5341 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5401 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     5461 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5521 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     5581 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5641 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     5701 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     5761 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     5821 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     5881 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     5941 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     6001 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     6061 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     6121 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     6181 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     6241 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     6301 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6361 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6421 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6481 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6541 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6601 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6661 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6721 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6781 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6841 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6901 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6961 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     7021 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7081 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     7141 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     7201 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     7261 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     7321 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7381 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7441 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7501 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7561 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7621 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7681 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     7741 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     7801 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaag
//
