LOCUS       sequence-492363-        6389 bp    DNA     circular SYN 10-NOV-2025
DEFINITION  Vector containing a Clover-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 6389)
  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 6389)
  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(Clover)-ScarlessDsRed
FEATURES             Location/Qualifiers
     source          1..6389
                     /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"
     primer_bind     complement(1919..1940)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2527..2548
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   3351..3413
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     regulatory      3808..3817
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3814..4491
                     /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"
     primer_bind     complement(3944..3962)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(3994..4014)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     4212..4231
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4406..4429
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4612..4733
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4649..4668)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4703..4722
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5008..5042
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5338..5357)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5363..5382)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        5424..5515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5516..5584,5585..6376)
                     /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(5734..5753)
                     /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 ggagctgttc accggggttg tgcccatcct
     1921 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtccgcggcg agggcgaggg
     1981 cgatgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     2041 gccctggccc accctcgtga ccaccttcgg ctacggcgtg gcctgcttca gccgctaccc
     2101 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     2161 gcgcaccatc tctttcaagg acgacggtac ctacaagacc cgcgccgagg tgaagttcga
     2221 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     2281 catcctgggg cacaagctgg agtacaactt caacagccac aacgtctata tcacggccga
     2341 caagcagaag aacggcatca aggctaactt caagatccgc cacaacgttg aggacggcag
     2401 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     2461 gcccgacaac cactacctga gccatcagtc cgccctgagc aaagacccca acgagaagcg
     2521 cgatcacatg gtcctgctgg agttcgtgac cgccgccggc tccggctctc cagaccccgc
     2581 ggcgcacctg cccttcttct acggcagcat ctcgcgtgcc gaggccgagg agcacctgaa
     2641 gctggcgggc atggcggacg ggctcttcct gctgcgccag tgcctgcgct cgctgggcgg
     2701 ctatgtgctg tcgctcgtgc acgatgtgcg cttccaccac tttcccatcg agcgccagct
     2761 caacggcacc tacgccattg ccggcggcaa agcgcactgt ggaccggcag agctctgcga
     2821 gttctactcg cgcgaccccg acgggctgcc ctgcaacctg cgcaagccgt gcaaccggcc
     2881 gtcgggcctc gagccgcagc cgggggtctt cgactgcctg cgagacgcca tggtgcgtga
     2941 ctacgtgcgc cagacgtgga agctggaggg cgaggccctg gagcaggcca tcatcagcca
     3001 ggccccgcag gtggagaagc tcattgctac gacggcccac gagcggatgc cctggtacca
     3061 cagcagcctg acgcgtgagg aggccgagcg caaactttac tctggggcgc agaccgacgg
     3121 caagttcctg ctgaggccgc ggaaggagca gggcacatac gccctgtccc tcatctatgg
     3181 gaagacggtg taccactacc tcatcagcca agacaaggcg ggcaagtact gcattcccga
     3241 gggcaccaag tttgacacgc tctggcagct ggtggagtat ctgaagctga aggcggacgg
     3301 gctcatctac tgcctgaagg aggcctgccc caacagcagt gcctgattaa ccctagaaag
     3361 ataatcatat tgtgacgtac gttaaagata atcatgcgta aaattgacgc atgtgtttta
     3421 tcggtctgta tatcgaggtt tatttattaa tttgaataga tattaagttt tattatattt
     3481 acacttacat actaataata aattcaacaa acaatttatt tatgtttatt tatttattaa
     3541 aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa aacttttagg atctaattca
     3601 attagagact aattcaatta gagctaattc aattaggatc caagcttatc gatttcgaac
     3661 cctcgaccgc cggagtataa atagaggcgc ttcgtctacg gagcgacaat tcaattcaaa
     3721 caagcaaagt gaacacgtcg ctaagcgaaa gctaagcaaa taaacaagcg cagctgaaca
     3781 agctaaacaa tcggctcgaa gccggtcgcc accatggcct cctccgagga cgtcatcaag
     3841 gagttcatgc gcttcaaggt gcgcatggag ggctccgtga acggccacga gttcgagatc
     3901 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
     3961 aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcca gtacggctcc
     4021 aaggtgtacg tgaagcaccc cgccgacatc cccgactaca agaagctgtc cttccccgag
     4081 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag
     4141 gactcctccc tccaggacgg ctccttcatc tacaaggtga agttcatcgg cgtgaacttc
     4201 ccctccgacg gccccgtaat gcagaagaag actatgggct gggaggcgtc caccgagcgc
     4261 ctgtaccccc gcgacggcgt gctgaagggc gagatccaca aggccctgaa gctgaaggac
     4321 ggcggccact acctggtgga gttcaagtcc atctacatgg ccaagaagcc cgtgcagctg
     4381 cccggctact actacgtgga ctccaagctg gacatcacct cccacaacga ggactacacc
     4441 atcgtggagc agtacgagcg cgccgagggc cgccaccacc tgttcctgta gcggccgcga
     4501 ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     4561 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     4621 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4681 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttagatatct
     4741 ataacaagaa aatatatata taataagtta tcacgtaagt agaacatgaa ataacaatat
     4801 aattatcgta tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg tcattttgac
     4861 tcacgcggtc gttatagttc aaaatcagtg acacttaccg cattgacaag cacgcctcac
     4921 gggagctcca agcggcgact gagatgtcct aaatgcacag cgacggattc gcgctattta
     4981 gaaagagaga gcaatatttc aagaatgcat gcgtcaattt tacgcagact atctttctag
     5041 ggttaaggaa gagccgtcgc tcttccccgg gaaaagaaga ccatatacgt ctcctatagg
     5101 tctcgtatac tgcttcctat acgacatcac cgatggggaa cacgaagcga tccagccccc
     5161 cttattagcc cgggcgaaag gcccagtctt tcgactgagc ctttcgtttt atttgatgcc
     5221 tggcagttcc ctactctcgc atggggagtc cccacactac catcggcgct acggcgtttc
     5281 acttctgagt tcggcatggg gtcaggtggg accaccgcgc tactgccgcc aggcaaacaa
     5341 ggggtgttat gagccatatt caggtataaa tgggctcgcg ataatgttca gaattggtta
     5401 attggttgta acactgaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5461 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agaatatgag
     5521 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5581 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5641 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5701 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5761 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5821 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5881 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5941 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6001 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6061 agcgatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6121 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6181 ccttccggct ggctggttta ttgctgataa atccggagcc ggtgagcgtg gttctcgcgg
     6241 tatcatcgca gcgctggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6301 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6361 gattaagcat tggtaagcag agcattacg
//
