LOCUS       sequence-492362-        6401 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  Vector containing a Scarlet-based pYtag biosensor for CRISPR 
            insertion into Drosophila RTKs, with a DsRed marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6401)
  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 6401)
  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-pYtag(Scarlet)-ScarlessDsRed
FEATURES             Location/Qualifiers
     source          1..6401
                     /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             1817..1873
                     /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             1874..2569
                     /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   3363..3425
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     regulatory      3820..3829
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3826..4503
                     /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    4624..4745
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4661..4680)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4715..4734
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5020..5054
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5350..5369)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5375..5394)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        5436..5527
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5528..5596,5597..6388)
                     /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(5746..5765)
                     /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 ggggccgagg
     1501 ctctggttct ccacctcccg tccctaatcc cgattatgag cccattcgga aaggtcaaag
     1561 agatttgtat agtggcctca atcaaagaag aatcgggtca ggttccggcc cgccgccagt
     1621 cccaaacccc gactatgaac caatcaggaa aggccaacgg gatttgtact caggattgaa
     1681 tcagcgcagg ataggttccg gctccgggcc accacccgtc cctaacccag attacgaacc
     1741 catacgcaag ggacaacgcg atctttacag cggattgaac caacgacgaa taggatctgg
     1801 ttctggcgga agcggagcta ctaacttcag cctgctgaag caggctggag acgtggagga
     1861 gaaccctgga cctatggtga gcaagggcga ggcagtgatc aaggagttca tgcggttcaa
     1921 ggtgcacatg gagggctcca tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     1981 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
     2041 cttctcctgg gacatcctgt cccctcagtt catgtacggc tccagggcct tcatcaagca
     2101 ccccgccgac atccccgact actataagca gtccttcccc gagggcttca agtgggagcg
     2161 cgtgatgaac ttcgaggacg gcggcgccgt gaccgtgacc caggacacct ccctggagga
     2221 cggcaccctg atctacaagg tgaagctccg cggcaccaac ttccctcctg acggccccgt
     2281 aatgcagaag aagacaatgg gctgggaagc gtccaccgag cggttgtacc ccgaggacgg
     2341 cgtgctgaag ggcgacatta agatggccct gcgcctgaag gacggcggcc gctacctggc
     2401 ggacttcaag accacctaca aggccaagaa gcccgtgcag atgcccggcg cctacaacgt
     2461 cgaccgcaag ttggacatca cctcccacaa cgaggactac accgtggtgg aacagtacga
     2521 acgctccgag ggccgccact ccaccggcgg catggacgag ctgtacaagg gctccggctc
     2581 tccagacccc gcggcgcacc tgcccttctt ctacggcagc atctcgcgtg ccgaggccga
     2641 ggagcacctg aagctggcgg gcatggcgga cgggctcttc ctgctgcgcc agtgcctgcg
     2701 ctcgctgggc ggctatgtgc tgtcgctcgt gcacgatgtg cgcttccacc actttcccat
     2761 cgagcgccag ctcaacggca cctacgccat tgccggcggc aaagcgcact gtggaccggc
     2821 agagctctgc gagttctact cgcgcgaccc cgacgggctg ccctgcaacc tgcgcaagcc
     2881 gtgcaaccgg ccgtcgggcc tcgagccgca gccgggggtc ttcgactgcc tgcgagacgc
     2941 catggtgcgt gactacgtgc gccagacgtg gaagctggag ggcgaggccc tggagcaggc
     3001 catcatcagc caggccccgc aggtggagaa gctcattgct acgacggccc acgagcggat
     3061 gccctggtac cacagcagcc tgacgcgtga ggaggccgag cgcaaacttt actctggggc
     3121 gcagaccgac ggcaagttcc tgctgaggcc gcggaaggag cagggcacat acgccctgtc
     3181 cctcatctat gggaagacgg tgtaccacta cctcatcagc caagacaagg cgggcaagta
     3241 ctgcattccc gagggcacca agtttgacac gctctggcag ctggtggagt atctgaagct
     3301 gaaggcggac gggctcatct actgcctgaa ggaggcctgc cccaacagca gtgcctgatt
     3361 aaccctagaa agataatcat attgtgacgt acgttaaaga taatcatgcg taaaattgac
     3421 gcatgtgttt tatcggtctg tatatcgagg tttatttatt aatttgaata gatattaagt
     3481 tttattatat ttacacttac atactaataa taaattcaac aaacaattta tttatgttta
     3541 tttatttatt aaaaaaaaac aaaaactcaa aatttcttct ataaagtaac aaaactttta
     3601 ggatctaatt caattagaga ctaattcaat tagagctaat tcaattagga tccaagctta
     3661 tcgatttcga accctcgacc gccggagtat aaatagaggc gcttcgtcta cggagcgaca
     3721 attcaattca aacaagcaaa gtgaacacgt cgctaagcga aagctaagca aataaacaag
     3781 cgcagctgaa caagctaaac aatcggctcg aagccggtcg ccaccatggc ctcctccgag
     3841 gacgtcatca aggagttcat gcgcttcaag gtgcgcatgg agggctccgt gaacggccac
     3901 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     3961 ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc
     4021 cagtacggct ccaaggtgta cgtgaagcac cccgccgaca tccccgacta caagaagctg
     4081 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
     4141 accgtgaccc aggactcctc cctccaggac ggctccttca tctacaaggt gaagttcatc
     4201 ggcgtgaact tcccctccga cggccccgta atgcagaaga agactatggg ctgggaggcg
     4261 tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca caaggccctg
     4321 aagctgaagg acggcggcca ctacctggtg gagttcaagt ccatctacat ggccaagaag
     4381 cccgtgcagc tgcccggcta ctactacgtg gactccaagc tggacatcac ctcccacaac
     4441 gaggactaca ccatcgtgga gcagtacgag cgcgccgagg gccgccacca cctgttcctg
     4501 tagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct
     4561 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     4621 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4681 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4741 tcttagatat ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg
     4801 aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg
     4861 cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca
     4921 agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat
     4981 tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga
     5041 ctatctttct agggttaagg aagagccgtc gctcttcccc gggaaaagaa gaccatatac
     5101 gtctcctata ggtctcgtat actgcttcct atacgacatc accgatgggg aacacgaagc
     5161 gatccagccc cccttattag cccgggcgaa aggcccagtc tttcgactga gcctttcgtt
     5221 ttatttgatg cctggcagtt ccctactctc gcatggggag tccccacact accatcggcg
     5281 ctacggcgtt tcacttctga gttcggcatg gggtcaggtg ggaccaccgc gctactgccg
     5341 ccaggcaaac aaggggtgtt atgagccata ttcaggtata aatgggctcg cgataatgtt
     5401 cagaattggt taattggttg taacactgac ccctatttgt ttatttttct aaatacattc
     5461 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5521 gaagaatatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5581 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5641 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5701 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5761 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5821 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5881 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5941 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     6001 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     6061 cacgatgcct gtagcgatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     6121 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     6181 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatccggag ccggtgagcg
     6241 tggttctcgc ggtatcatcg cagcgctggg gccagatggt aagccctccc gtatcgtagt
     6301 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6361 aggtgcctca ctgattaagc attggtaagc agagcattac g
//
