LOCUS       sequence-423480-        6253 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses equimolar Peroxisome- and Endoplasmic Reticulum- targeted 
            split-FAST fragments for Short-range ER-PO Contact sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6253)
  AUTHORS   Cali T
  TITLE     SPLICSFAST-P2A
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6253)
  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            "Unpublished"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-ER-PO-P2A
FEATURES             Location/Qualifiers
     source          1..6253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(23..42)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     77..96
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(156..172)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(156..172)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(169..191)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    180..196
                     /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(204..210,211..228,229..234))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    249..270
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(387..404)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(558..1146)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(638..657)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1317..2108,2109..2177))
                     /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     1940..1959
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2178..2282)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2357..2376)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2548..2927
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2928..3131
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3082..3102
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3176..3195
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3176..3194
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3214..3223
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3514..3537
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3565..3621
                     /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(4157..4174)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4163..4387
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4433..4861
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4520..4539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4730..4751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4870..4890)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4875..5204
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5055..5190
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5117..5136
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5271..6065
                     /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(5325..5344)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5935..5954
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    join(6239..6253,1..107)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
       61 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
      121 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
      181 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
      241 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
      301 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
      361 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
      421 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
      481 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
      541 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
      601 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
      661 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
      721 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
      781 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
      841 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
      901 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
      961 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     1021 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     1081 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     1141 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     1201 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     1261 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     1321 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     1381 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     1441 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     1501 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     1561 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     1621 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     1681 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     1741 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     1801 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     1861 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     1921 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     1981 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     2041 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     2101 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     2161 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     2221 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     2281 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     2341 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     2401 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
     2461 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
     2521 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
     2581 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     2641 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     2701 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     2761 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     2821 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     2881 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     2941 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     3001 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     3061 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     3121 taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata
     3181 cgactcacta tagggagacc caagctggct agcgccacca tgggcgacag ctactgggtg
     3241 ttcgtgaagc gggaattcgg cggcgacggc ggaagcggag gcggcgacat cagcaaactc
     3301 agagtgttcc tggctctgcc tatcattatg gtggtggcct ttagcatgtg catcatctgc
     3361 ctgctgatgg ccggagatac atggaccgag acactggctt atgtgctgtt ctggggcgtg
     3421 gcctctatcg gcacattctt catcatcctg tacaacggca aggacttcgt ggacgcccct
     3481 agactggtcc agaaggaaaa gatcgacgac atcgactaca aggacgacga cgataagctt
     3541 aaagagctgg cttgtggcag cggcgccacc aacttttctc tgctgaagca ggccggcgac
     3601 gtcgaggaaa accccggccc acggagcatg gaacacgtgg ccttcggcag tgaagatatc
     3661 gagaacaccc tggccaagat ggacgacgga caactggatg gcctggcctt tggcgccatt
     3721 cagctggacg gcgacggcaa catcctgcag tacaatgccg ccgagggcga tatcaccggc
     3781 agagatccta agcaggtgat cggaaaaaac ttctttaagg atgtggcccc tggaacagac
     3841 agccccgagt tctacggcaa gttcaaggag ggcgtggctt ctggcaatct gaacaccatg
     3901 ttcgagtgga tgatccccac cagcagaggc cctaccaagg tgaaagtgca catgaaaaag
     3961 gccctgagcg gcggcgatgg aggctccggc ggtggctcca agctgtggtg gcctttcgag
     4021 atgagccctg gagttctgac cttcgccatc atctggccct tcatcgccca gtggctggtg
     4081 tacctgtact accagagacg gagaaggaag ctcaactgac tcgagtctag agggcccgtt
     4141 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     4201 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     4261 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     4321 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     4381 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     4441 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4501 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4561 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4621 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     4681 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4741 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     4801 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4861 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     4921 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     4981 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     5041 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     5101 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     5161 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     5221 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     5281 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     5341 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     5401 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     5461 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     5521 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     5581 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     5641 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     5701 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     5761 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     5821 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     5881 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     5941 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     6001 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     6061 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     6121 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     6181 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     6241 cttgtttatt gca
//
