LOCUS       sequence-492360-        8420 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  Vector for tagging Breathless (Btl) with Scarlet-based pYtag 
            biosensor. DsRed marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8420)
  AUTHORS   Ho EK, Kim-Yip RP, Simpkins AG, Farahani PE, Oatman HR, Posfai E, 
            Shvartsman SY, Toettcher JE
  TITLE     A live-cell biosensor of in vivo receptor tyrosine kinase activity 
            reveals feedback regulation of a developmental gradient.
  JOURNAL   Cell Rep. 2025 Jul 22;44(7):115930. doi: 
            10.1016/j.celrep.2025.115930. Epub 2025 Jun 27.
  PUBMED    40581929
REFERENCE   2  (bases 1 to 8420)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 10, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep.";
            date: "2025-07-22"; volume: "44(7):115930. doi"; pages: " 
            10.1016/j.celrep.2025.115930. Epub 2025 Jun 27"
COMMENT     Sequence Label: pHD Btl-pYtag(Scarlet) ScarlessDsRed
FEATURES             Location/Qualifiers
     source          1..8420
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      109..697
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     598..617
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     851..868
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     966..983
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     967..983
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2817..2873
                     /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             2874..3569
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     repeat_region   4363..4425
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     regulatory      4820..4829
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4826..5503
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     polyA_signal    5624..5745
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5661..5680)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5715..5734
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6020..6054
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7369..7388)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(7394..7413)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        7455..7546
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7547..7615,7616..8407)
                     /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(7765..7784)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttacgcgcgc
       61 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
      121 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
      181 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
      241 gataccaaat actgttcttc tagtgtagcc gtagttagcc caccacttca agaactctgt
      301 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
      361 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
      421 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
      481 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
      541 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      601 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      661 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      721 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      781 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      841 gaccgagcgc agcgagtcag tgagcgagga agcggaaggc gagagtaggg aactgccagg
      901 catcaaacta agcagaaggc ccctgacgga tggccttttt gcgtttctac aaactctttc
      961 tgtgttgtaa aacgacggcc agtcttaagc tcgggccccc tgggcggttc tgataacgag
     1021 taatcgttaa tccgcaaata acgtaaaaac ccgcttcggc gggttttttt atggggggag
     1081 tttagggaaa gagcatttgt cagaatattt aagggcgcct gtcactttgc ttgatatatg
     1141 agaattattt aaccttataa atgagaaaaa agcaacgcac tttaaataag atacgttgct
     1201 ttttcgattg atgaacacct ataattaaac tattcatcta ttatttatga ttttttgtat
     1261 atacaatatt tctagtttgt taaagagaat taagaaaata aatctcgaaa ataataaagg
     1321 gaaaatcagt ttttgatatc aaaattatac atgtcaacga taatacaaaa tataatacaa
     1381 actataagat gttatcagta tttattatgc atttagaata cgtactcagc gtctgggttc
     1441 cccatcggtg atgtcgtata agagacgtat aggagaccta tagtgtcttc ggggccgaaa
     1501 gatggtcaag gaggagcaca cggatacgga catggccagt ttggtgcgtg aaatggaggt
     1561 gatgaaaatg attggcaagc acataaacat catcaatctg ctaggctgct gcagccaggg
     1621 cggtcctctg tgggtgatcg tggagtatgc tccacacgga aacctcaagg acttcctcaa
     1681 gcagaatcga cctggtgctc cacagcgacg gagtgacagt gatggctatc tagatgacaa
     1741 gccactgata cccactcaac atcttggcga aaaggagctc accaagtttg catttcaaat
     1801 cgctcgagga atggagtacc ttgcttcaag acgggtaagt tcatccaaga atgtctggct
     1861 tttaaaacct cccctcccat aactatttat aaagatactt ataactctta ctctttcagt
     1921 gcattcatcg cgatctggct gcccgtaatg ttcttgtaag cgatggatac gttatgaaaa
     1981 tcgccgattt tggtctggcg agggatattc aagatacgga gtactaccga aaaaatacca
     2041 atggccggtt gcccattaag tggatggcgc ccgagtcgct gcaggagaag aagtacgact
     2101 cacagagcga cgtctggagc tatggtgttc tgctgtggga gattatgacc tacggggatc
     2161 aaccataccc gcacatactg tccgccgagg agctctacag ctacctgatt acgggtcagc
     2221 gcatggagaa accggctaag tgctccctga acatctacgt ggtaatgcga cagtgctggc
     2281 actttgagtc ctgtgcacga cccacgttcg cggagctggt agagagtttc gatgggatcc
     2341 tgcagcaggc tagtagcaat ccgaacgatg cctatttgga cctttcgatg cccatgctgg
     2401 agacgccgcc ctcatcgggg gatgaggatg atggctccga caccgaaaca ttccgggaaa
     2461 cgtctccact gagatatcag tacacctata agtttaatgg ctctggttct ccacctcccg
     2521 tccctaatcc cgattatgag cccattcgga aaggtcaaag agatttgtat agtggcctca
     2581 atcaaagaag aatcgggtca ggttccggcc cgccgccagt cccaaacccc gactatgaac
     2641 caatcaggaa aggccaacgg gatttgtact caggattgaa tcagcgcagg ataggttccg
     2701 gctccgggcc accacccgtc cctaacccag attacgaacc catacgcaag ggacaacgcg
     2761 atctttacag cggattgaac caacgacgaa taggatctgg ttctggcgga agcggagcta
     2821 ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga cctatggtga
     2881 gcaagggcga ggcagtgatc aaggagttca tgcggttcaa ggtgcacatg gagggctcca
     2941 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     3001 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttctcctgg gacatcctgt
     3061 cccctcagtt catgtacggc tccagggcct tcatcaagca ccccgccgac atccccgact
     3121 actataagca gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     3181 gcggcgccgt gaccgtgacc caggacacct ccctggagga cggcaccctg atctacaagg
     3241 tgaagctccg cggcaccaac ttccctcctg acggccccgt aatgcagaag aagacaatgg
     3301 gctgggaagc gtccaccgag cggttgtacc ccgaggacgg cgtgctgaag ggcgacatta
     3361 agatggccct gcgcctgaag gacggcggcc gctacctggc ggacttcaag accacctaca
     3421 aggccaagaa gcccgtgcag atgcccggcg cctacaacgt cgaccgcaag ttggacatca
     3481 cctcccacaa cgaggactac accgtggtgg aacagtacga acgctccgag ggccgccact
     3541 ccaccggcgg catggacgag ctgtacaagg gctccggctc tccagacccc gcggcgcacc
     3601 tgcccttctt ctacggcagc atctcgcgtg ccgaggccga ggagcacctg aagctggcgg
     3661 gcatggcgga cgggctcttc ctgctgcgcc agtgcctgcg ctcgctgggc ggctatgtgc
     3721 tgtcgctcgt gcacgatgtg cgcttccacc actttcccat cgagcgccag ctcaacggca
     3781 cctacgccat tgccggcggc aaagcgcact gtggaccggc agagctctgc gagttctact
     3841 cgcgcgaccc cgacgggctg ccctgcaacc tgcgcaagcc gtgcaaccgg ccgtcgggcc
     3901 tcgagccgca gccgggggtc ttcgactgcc tgcgagacgc catggtgcgt gactacgtgc
     3961 gccagacgtg gaagctggag ggcgaggccc tggagcaggc catcatcagc caggccccgc
     4021 aggtggagaa gctcattgct acgacggccc acgagcggat gccctggtac cacagcagcc
     4081 tgacgcgtga ggaggccgag cgcaaacttt actctggggc gcagaccgac ggcaagttcc
     4141 tgctgaggcc gcggaaggag cagggcacat acgccctgtc cctcatctat gggaagacgg
     4201 tgtaccacta cctcatcagc caagacaagg cgggcaagta ctgcattccc gagggcacca
     4261 agtttgacac gctctggcag ctggtggagt atctgaagct gaaggcggac gggctcatct
     4321 actgcctgaa ggaggcctgc cccaacagca gtgcctgatt aaccctagaa agataatcat
     4381 attgtgacgt acgttaaaga taatcatgcg taaaattgac gcatgtgttt tatcggtctg
     4441 tatatcgagg tttatttatt aatttgaata gatattaagt tttattatat ttacacttac
     4501 atactaataa taaattcaac aaacaattta tttatgttta tttatttatt aaaaaaaaac
     4561 aaaaactcaa aatttcttct ataaagtaac aaaactttta ggatctaatt caattagaga
     4621 ctaattcaat tagagctaat tcaattagga tccaagctta tcgatttcga accctcgacc
     4681 gccggagtat aaatagaggc gcttcgtcta cggagcgaca attcaattca aacaagcaaa
     4741 gtgaacacgt cgctaagcga aagctaagca aataaacaag cgcagctgaa caagctaaac
     4801 aatcggctcg aagccggtcg ccaccatggc ctcctccgag gacgtcatca aggagttcat
     4861 gcgcttcaag gtgcgcatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     4921 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg
     4981 ccccctgccc ttcgcctggg acatcctgtc cccccagttc cagtacggct ccaaggtgta
     5041 cgtgaagcac cccgccgaca tccccgacta caagaagctg tccttccccg agggcttcaa
     5101 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     5161 cctccaggac ggctccttca tctacaaggt gaagttcatc ggcgtgaact tcccctccga
     5221 cggccccgta atgcagaaga agactatggg ctgggaggcg tccaccgagc gcctgtaccc
     5281 ccgcgacggc gtgctgaagg gcgagatcca caaggccctg aagctgaagg acggcggcca
     5341 ctacctggtg gagttcaagt ccatctacat ggccaagaag cccgtgcagc tgcccggcta
     5401 ctactacgtg gactccaagc tggacatcac ctcccacaac gaggactaca ccatcgtgga
     5461 gcagtacgag cgcgccgagg gccgccacca cctgttcctg tagcggccgc gactctagat
     5521 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     5581 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     5641 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     5701 actgcattct agttgtggtt tgtccaaact catcaatgta tcttagatat ctataacaag
     5761 aaaatatata tataataagt tatcacgtaa gtagaacatg aaataacaat ataattatcg
     5821 tatgagttaa atcttaaaag tcacgtaaaa gataatcatg cgtcattttg actcacgcgg
     5881 tcgttatagt tcaaaatcag tgacacttac cgcattgaca agcacgcctc acgggagctc
     5941 caagcggcga ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt tagaaagaga
     6001 gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga ctatctttct agggttaatt
     6061 cgtagtataa ggagaccaaa aagaattcca acgagtcaat cagatcccat cgaagcatcg
     6121 ccaaccatca tctcatccca gaaagtgcca aagaaaagat cgcagaagca gaaacatcag
     6181 aacaagtttt tcaaactacc acgaaaatta agctctaaac ttcgcattgt ttgaattggt
     6241 gtatttaata aataataaat ccgatatcag atatccaatg ctacaagaac tacgcgattt
     6301 gggcttgatt acaaagactt aacttgcact tagcaaactc attctttagt tttacaaagt
     6361 tcatatgtat ctttgtatat ctacaatgtt ttgttctggg tctttgggct acatttattt
     6421 catagtgttt cttgttgaac caataaaaac gaatttgttt tactgtaact aagaattttg
     6481 atcaccacaa ataatgctca ctttctccca agaaccgctc ataatcaacc gcagagataa
     6541 ccaagttcag tgtgttttat atgaaaacaa aacaagagag caagctagct gtggcaattc
     6601 caagcttata tacccttgca gtaattctat atttatacga aaaagaagac tttatgataa
     6661 aaaaacacgg aaatgtgata tcttttctta ttacccgaaa tatatagaag tttttaggaa
     6721 aagtggcagg catttatata ctagtttacg tagaaatcaa tgcttcctta gtaagggtat
     6781 gaaacaaagc aatcgcattt aagaatatat gcagaagtca gttcatgtca tcgatcagtc
     6841 tgcgtcaaaa tgttaagatt aacgtttgtg tgcctttgtt ttctcgttct tcaaaataga
     6901 acagaaagtt ttggcagtcc ttggaagtat ttcggatcat caaatgtaga agtgaattat
     6961 aatcaaaggc aagatcgtca gtattttctc ctgaatttgc tcattcaagt ccacaaacct
     7021 ttgcttcacg aggaactcat tacaatgggg agccaactta acgatgatcc tgacagcgga
     7081 agagccgtcg ctcttccccg ggaaaagaag accatatacg tctcctatag gtctcgtata
     7141 ctgcttccta tacgacatca ccgatgggga acacgaagcg atccagcccc ccttattagc
     7201 ccgggcgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggcagttc
     7261 cctactctcg catggggagt ccccacacta ccatcggcgc tacggcgttt cacttctgag
     7321 ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc caggcaaaca aggggtgtta
     7381 tgagccatat tcaggtataa atgggctcgc gataatgttc agaattggtt aattggttgt
     7441 aacactgacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     7501 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagaatatga gtattcaaca
     7561 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     7621 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     7681 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     7741 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     7801 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     7861 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     7921 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     7981 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     8041 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcgatggc
     8101 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     8161 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     8221 tggctggttt attgctgata aatccggagc cggtgagcgt ggttctcgcg gtatcatcgc
     8281 agcgctgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     8341 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     8401 ttggtaagca gagcattacg
//
