LOCUS       sequence-362561-        8287 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-specific donor plasmid for intergrating a transcription unit 
            for expression of ASAP2f voltage reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8287)
  AUTHORS   Blanch-Asensio A, Grandela C, Brandao KO, de Korte T, Mei H, 
            Ariyurek Y, Yiangou L, Mol MPH, van Meer BJ, Kloet SL, Mummery CL, 
            Davis RP
  TITLE     STRAIGHT-IN enables high-throughput targeting of large DNA payloads 
            in human pluripotent stem cells.
  JOURNAL   Cell Rep Methods. 2022 Sep 22;2(10):100300. doi: 
            10.1016/j.crmeth.2022.100300. eCollection 2022 Oct 24.
  PUBMED    36313798
REFERENCE   2  (bases 1 to 8287)
  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: "Cell Rep 
            Methods."; date: "2022-09-22"; volume: "2(10):100300. doi"; pages: "
            10.1016/j.crmeth.2022.100300. eCollection 2022 Oct 24"
COMMENT     Sequence Label: Bxb1-ASAP2f
FEATURES             Location/Qualifiers
     source          1..8287
                     /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        1869..2248
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          2527..3543
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3467..3489
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3551..3570
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      3703..3712
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(5104..5121)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5110..5334
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(5376..5408)
                     /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(5416..5433)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5416..5432)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5425..5447)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5645..6100
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5732..5751)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5942..5963
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6117..6136)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6236..6258
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6296..6314)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6382..6486
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6487..6555,6556..7347)
                     /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(6705..6724)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7518..8106
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8007..8026
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8260..8277
                     /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 acggggtcga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     1921 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     1981 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     2041 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     2101 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     2161 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     2221 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
     2281 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
     2341 tgcagcgatg ggggcggggg gggggggnnn sgsgcgccrg gsggggsggg gsggggsgrg
     2401 gggsggggsg gggsgrggsg grgrggkgsg gsggcagcca atcagagcgg cgcgctccga
     2461 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
     2521 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
     2581 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
     2641 tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg
     2701 cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt
     2761 gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt
     2821 gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg
     2881 gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg
     2941 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc
     3001 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg
     3061 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggkggg ggtgccgggc
     3121 ggggcggggc cgccycgggc cggggrgggc tcgggggrgg ggcgcggcgg cccccggagc
     3181 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag
     3241 agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc
     3301 cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
     3361 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc
     3421 tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg
     3481 cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta
     3541 cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattcctc
     3601 gaggaattca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg tgtggccaat
     3661 gccctggctc acaaatacca ctgagatctg catgagctag ccgccaccat ggagacgact
     3721 gtgaggtatg aacaggggtc agagctcact aaaacttcga gctctccaac agcagatgag
     3781 cccacgataa agattgatga tggtcgtgat gagggtaatg aacaagacag ctgttccaat
     3841 accattagga gaaaaatttc cccgtttgtg atgtcatttg gattcagagt atttggagtt
     3901 gtgcttatca ttgtagacat catagtggtg attgtggatc tggccatcag tgagaagaaa
     3961 agaggcatta gagagattct tgaaggtgtt tccctggcta tagcactctt cttccttgtt
     4021 gatgttctca tgagagtgtt tgttgaaggc ttcaagaact atttccggtc caaactgaat
     4081 actttggatg cagtcatagt agtgggcact ctgctaatta atatgaccta ctccttctct
     4141 gaccttagct ttaacagcca taacgtgtat attaccgcgg ataaacagaa aaacggcatt
     4201 aaagcgaact ttaccgtgcg ccataacgtg gaagatggca gcgtgcagct ggcggatcat
     4261 tatcagcaga acaccccgat tggcgatggc ccggtgctgc tgccggataa ccattatctg
     4321 agcacccaga ccgtgctgag caaagatccg aacgaaaaac gcgatcacat ggtgctgctg
     4381 gaatttgtga ccgcagcggg cattacacac ggcatggatg aactgtatgg cggcaccggc
     4441 ggcagcgcga gccagggcga agaactgttt accggcgtgg tgccgattct ggtggaactg
     4501 gatggcgatg tgaacggcca taaatttagc gtgcgcggcg aaggcgaagg cgatgcgacc
     4561 attggcaaac tgaccctgaa atttatttgc accaccggca aactaccggt gccgtggccg
     4621 accctggtga ccaccttaac ctatggcgtg cagtgcttta gccgctatcc ggatcatatg
     4681 aaacgccatg atttttttaa aagcgcgatg ccggaaggct atgtgcagga acgcaccatt
     4741 agctttaaag atgatggcaa atataaaacc cgcgcggtgg tgaaatttga aggcgatacc
     4801 ctggtgaacc gcattgaact gaaaggcacc gattttaaag aagatggcaa cattctgggg
     4861 cataaactgg aatataacac agatcagatg ccgcagatgg ttactcttct tcgagttctg
     4921 agaattgtta tcttaataag aatatttcgc ctggcttcac agaagaaaca acttgaagtg
     4981 gtaacctaat gaaagcttgg taccgagctc ggatccacta gtccagtgtg gtggaattct
     5041 gcagatatcc agcacagtgg cggccgctcg agtctagagg gcccgtttaa acccgctgat
     5101 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     5161 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     5221 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     5281 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     5341 aggcggaact agcgctgcaa gaactcttcc tcacgataac ttcgtatagg agactttata
     5401 cgaagttaag cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     5461 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     5521 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat
     5581 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     5641 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     5701 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     5761 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     5821 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     5881 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5941 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     6001 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     6061 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     6121 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     6181 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     6241 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     6301 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     6361 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     6421 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     6481 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     6541 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     6601 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     6661 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     6721 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     6781 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     6841 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     6901 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     6961 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     7021 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     7081 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     7141 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     7201 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     7261 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     7321 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     7381 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     7441 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     7501 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     7561 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     7621 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     7681 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     7741 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     7801 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     7861 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     7921 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     7981 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     8041 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     8101 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     8161 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     8221 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     8281 gcggaag
//
