LOCUS       sequence-423486-        6145 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses equimolar Peroxisome- and Mitochondria- targeted 
            split-FAST fragments for Short-range PO-MT Contact sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6145)
  AUTHORS   Cali T
  TITLE     SPLICSFAST-P2A
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6145)
  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: 
            "Unpublished"
COMMENT     Sequence Label: SPLICSFAST-PO-MT-P2A
FEATURES             Location/Qualifiers
     source          1..6145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1114..1170
                     /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             1180..1209
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1736..1753)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1742..1966
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2012..2440
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2099..2118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2309..2330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2449..2469)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2454..2783
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2634..2769
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2696..2715
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2850..3644
                     /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(2904..2923)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3514..3533
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3818..3939
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3855..3874)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3909..3928
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3988..4004)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3988..4004)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4001..4023)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4012..4028
                     /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(4036..4042,4043..4060,4061..4066))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4081..4102
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4219..4236)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4390..4978)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4470..4489)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5149..5940,5941..6009))
                     /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     5772..5791
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6010..6114)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccaccatgg gcgatagcta ctgggtgttc gtgaaaagag gcggcgacgg cggcagcgga
      961 ggtggctcta agctgtggtg gccttttgag atgagccctg gcgtgctgac cttcgccatt
     1021 atctggccat tcatcgccca gtggctggtg tacctgtact accagagacg gagaaggaag
     1081 ctcaacctga aagagctggc ttgtggctct ggcgctacaa acttcagcct gctgaagcag
     1141 gccggcgacg tggaagagaa ccccggccct agaagcatgg aacagaagct gatctctgaa
     1201 gaggatctgg aattcgccgc tatggtgggc cggaactccg ccatcgccgc aggcgtctgc
     1261 ggcgccctgt tcatcggcta ctgcatctat ttcgacagaa agcggcggag cgaccctaac
     1321 agcggaggcg gtggatctgg tggtggcgga tcaatggaac acgtggcctt cggctctgag
     1381 gacatcgaga acacactggc caagatggac gacggccagc tggacggcct ggcctttggc
     1441 gccatccagc tggatggcga tggaaatatc ctgcagtaca acgccgccga gggcgacatc
     1501 accggcagag atcctaagca agtgatcggc aagaactttt tcaaggacgt ggcccctgga
     1561 acagacagcc ccgagttcta cggcaaattc aaggaaggcg ttgcttctgg caatctgaac
     1621 accatgttcg agtggatgat ccccaccagc agaggaccta ccaaggtgaa ggtgcacatg
     1681 aaaaaggccc tgagctgact cgagtctaga gggcccgttt aaacccgctg atcagcctcg
     1741 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     1801 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     1861 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     1921 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     1981 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     2041 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2101 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2161 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2221 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2281 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2341 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2401 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     2461 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2521 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2581 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     2641 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     2701 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     2761 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     2821 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     2881 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     2941 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3001 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3061 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3121 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3181 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3241 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3301 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3361 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3421 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3481 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3541 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3601 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     3661 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     3721 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     3781 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     3841 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3901 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     3961 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4021 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4081 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4141 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4201 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4261 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4321 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4381 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4441 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4501 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4561 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4621 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4681 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4741 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4801 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4861 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4921 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4981 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5041 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5101 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5161 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5221 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5281 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5341 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5401 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5461 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5521 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5581 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5641 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5701 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5761 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5821 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5881 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5941 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6001 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6061 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6121 tccccgaaaa gtgccacctg acgtc
//
