LOCUS       sequence-350004-        6637 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for knock-in of hsp70-EGFP using CRISPR-Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6637)
  AUTHORS   Wucherpfennig JI, Howes TR, Au JN, Au EH, Roberts Kingman GA, Brady 
            SD, Herbert AL, Reimchen TE, Bell MA, Lowe CB, Dalziel AC, Kingsley 
            DM
  TITLE     Evolution of stickleback spines through independent cis-regulatory 
            changes at HOXDB.
  JOURNAL   Nat Ecol Evol. 2022 Sep 1. pii: 10.1038/s41559-022-01855-3. doi: 
            10.1038/s41559-022-01855-3.
  PUBMED    36050398
REFERENCE   2  (bases 1 to 6637)
  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 Ecol 
            Evol. 2022 Sep 1. pii: 10.1038/s41559-022-01855-3. doi: 
            10.1038/s41559-022-01855-3."
COMMENT     Sequence Label: ptia1l-hsp-GFP
FEATURES             Location/Qualifiers
     source          1..6637
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1809..1818
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1815..1817,1818..1820,1821..2534)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1815..1817,1818..1820,1821..2531)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1860..1881)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2121..2140)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2468..2489
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2550..2684
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2574..2593)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2628..2647
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2869..2885)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2869..2885)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2882..2904)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2893..2909
                     /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)."
     promoter        complement(join(2917..2923,2924..2941,2942..2947))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2962..2983
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3100..3117)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3271..3859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3351..3370)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4030..4821,4822..4890))
                     /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     4653..4672
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(4908..5702)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5019..5038)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5629..5648
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(6046..6474)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6156..6177)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6368..6387
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6600..6622
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6614..6631
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6615..6631
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg cgaattgggc cctctagatg catgctcgag cggccgccag
       61 tgtgatggat atctgcagaa ttcgcccttg aattcggtat gtcgggaacc tctccaggga
      121 aaagcctctc acaggcagcc gatgcgggaa gaggtgtatt agtcttgata gagaggctgc
      181 aaatagcagg aaacgtgagc agagactccc tggtgtctga aacacaggcc agataggccc
      241 cttacgtacg ctagcgggcc cgatatcacc gggggatcct tcaggggtgt cgcttggtta
      301 tttccaaaaa tcaaattaat tttattaaac tattagaacg agcatgtttt gtctatatgc
      361 tacagaagat aaaaaataat aggagttaac agttataaaa caacacactt tgtttctatt
      421 gattgttgac cacactgggg tctcattaag ttagattaaa gacacactaa ctgggtcaaa
      481 agcagcagat tgatttcata gcaccagggt aaactttcta acacttttac ggcaatcata
      541 tacattaaaa ttaaatacag accacgactg aacaaggagg atgatctcca atattaaaca
      601 aagggacttg tgcctatttc tctgagggta aacatgacct ctcaagttag caagttgttt
      661 ttaacactac aaaaatagtt aagactgcaa tcccagaata aagtattggt tttaaccaat
      721 caatatagta cagtaaacat ccatttgttt tgttgaaacg ttaaacaaat ctgaccaaag
      781 ctattagctt atataaaaca ggtttgcctt ctatgtagct gaaaacacca caggcccgat
      841 tttgctactg tgtaaaacat ttcagcaaga tttttttatt gcattttttt ttactgaatc
      901 gttcaaacat tttatcattt tagtttgttc attcattgca actggaaaaa caacacatca
      961 cacaaccgca catatttcag caataagtac aataaaacac tcaaataaaa aaaacatttt
     1021 aaatctcttt gtatttttga ccgctgtttc gcgtaatttc acggtaaaac tctggaaatc
     1081 tccactacat tcctctcagc ggctcctctc aatgacagct gaagaagtga cgcggctgcc
     1141 tgctgtgttt tgattggtcg aattcactgg aggcttccag aacagtgtag agtctgaacg
     1201 ggtgcgcgct ctgctgtatt taaagggcga aagagagacc gcagagaaac tcaaccgaag
     1261 agaagcgact tgacaaagaa gaaaagagca gcctgacagg actttttccc cgacgaggtg
     1321 tttattcgct ctatttaaga atctactgta aggtaagtct caatatattg tactctattg
     1381 gctaatcaga attatataga gattatatgt acttaatgtc aaaaaatcaa ctttgtatat
     1441 gtaatctttt tacatgtgga ctgcctatgt tcatcttatt ttaggtctac tagaaaatta
     1501 tatttcccgt tttcacaata aggattttta aaaaaagcaa tgaacagacg ggcatttact
     1561 ttatgttgct gacattattt tacatgagca taataaccat aaatactagc aaatgtccta
     1621 aatgaatttg tgttaatgtt gtctacaaaa gaaaattagc gttttacttg tacaactaat
     1681 aataacttgg ttattaagag aatttcactt gttgactaga aaaatccttt cataatgaaa
     1741 caattgcacc ataaattgta taaatataaa attaattcta attgtttttt ttcctgcagt
     1801 cgacggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     1861 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     1921 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     1981 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     2041 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     2101 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     2161 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     2221 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     2281 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     2341 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     2401 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     2461 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     2521 agctgtacaa gtaaagcgac atcgatgatg atccagacat gataagatac attgatgagt
     2581 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     2641 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     2701 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
     2761 tctacaaatg tggtatggct gattatgatc ctctaaaggg cgaattccag cacactggcg
     2821 gccgttacta gtggatccga gctcggtacc aagcttggcg taatcatggt catagctgtt
     2881 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     2941 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     3001 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3061 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     3121 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3181 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3241 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3301 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3361 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3421 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3481 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3541 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3601 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3661 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3721 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3781 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3841 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3901 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3961 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4021 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4081 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     4141 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     4201 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     4261 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4321 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4381 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4441 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4501 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4561 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4621 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4681 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4741 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4801 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4861 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaattca gaagaactcg
     4921 tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg
     4981 aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct
     5041 atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg
     5101 ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg
     5161 ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc
     5221 tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg
     5281 atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc
     5341 cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtggga tgacaggaga
     5401 tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg
     5461 agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc
     5521 tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc
     5581 gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag
     5641 ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc
     5701 atgcgaaacg atcctcatcc tgtctcttga tcagatcttg atcccctgcg ccatcagatc
     5761 cttggcggca agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc
     5821 gccccagctg gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc
     5881 catgtaagcc cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag
     5941 atagcccagt agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac
     6001 gtgttccgct tcctttagca gcccttgcgc cctgaatttt gttaaaattc gcgttaaatt
     6061 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     6121 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     6181 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     6241 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     6301 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     6361 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     6421 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc
     6481 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     6541 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     6601 ccagtcacga cgttgtaaaa cgacggccag tgaattg
//
