LOCUS       sequence-423478-        6283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses equimolar Outer mitochondrial membrane- and Endoplasmic 
            Reticulum- targeted split-FAST fragments for Short-range ER-Mito 
            Contact sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6283)
  AUTHORS   Cali T
  TITLE     SPLICSFAST-P2A
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6283)
  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            "Unpublished"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-MT-P2A
FEATURES             Location/Qualifiers
     source          1..6283
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(72..91)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(751..1542,1543..1611))
                     /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     1374..1393
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1612..1716)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1791..1810)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1982..2361
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2362..2565
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2516..2536
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2610..2629
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2610..2628
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2648..2657
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2948..2971
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2999..3055
                     /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             3065..3094
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3621..3638)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3627..3851
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3897..4325
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3984..4003)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4194..4215
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4334..4354)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4339..4668
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4519..4654
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4581..4600
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4735..5529
                     /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(4789..4808)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5399..5418
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5703..5824
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5740..5759)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5794..5813
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5873..5889)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5873..5889)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5886..5908)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5897..5913
                     /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(5921..5927,5928..5945,5946..5951))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5966..5987
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6104..6121)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6275..6283,1..580))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
       61 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
      121 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
      181 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
      241 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
      301 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
      361 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
      421 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
      481 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
      541 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
      601 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
      661 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
      721 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
      781 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
      841 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
      901 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
      961 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     1021 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     1081 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     1141 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     1201 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     1261 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     1321 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     1381 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     1441 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     1501 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     1561 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     1621 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     1681 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     1741 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     1801 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     1861 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     1921 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     1981 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
     2041 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     2101 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     2161 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     2221 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     2281 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     2341 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     2401 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     2461 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     2521 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga
     2581 gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct
     2641 ggctagcgcc accatgggcg acagctactg ggtgttcgtg aaacgggagt ttggaggcga
     2701 cggaggatcc ggcggcgggg atatttctaa gctgagagtg ttcctggccc tgcctatcat
     2761 catggtcgtc gcttttagca tgtgcatcat ttgcctgctc atggccggag acacctggac
     2821 cgagacactg gcctacgtgc tgttttgggg cgttgccagc atcggcacct tcttcatcat
     2881 cctttataac ggcaaagact tcgtggacgc ccctcggctg gtgcagaaag agaagatcga
     2941 cgacatcgac tacaaggacg acgatgataa gctgaaagag ctggcctgtg gcagcggtgc
     3001 tacaaacttc tccctgctga agcaggccgg cgacgtggaa gaaaaccccg gccctagaag
     3061 catggaacag aagctgatca gcgaagagga cctggaattc gccgctatgg tgggccggaa
     3121 cagcgccatc gccgctggcg tgtgcggcgc cctgttcatc ggctactgca tctacttcga
     3181 cagaaagcgc agatctgatc ccaacagcgg tggcggcggc tccggcggcg gaggaagcat
     3241 ggaacacgtg gcctttggca gcgaggatat cgagaatacc ctggcaaaga tggacgacgg
     3301 ccagctggac ggcctggcct tcggcgctat ccagctggac ggcgatggca acatcctgca
     3361 gtacaacgcc gccgagggag atatcaccgg cagagatcct aagcaagtga tcggaaagaa
     3421 cttcttcaag gacgtggccc ctggcacaga cagccccgag ttctacggca agttcaagga
     3481 aggagtggcc tctggcaatc tgaacaccat gttcgagtgg atgatcccta cctctagagg
     3541 cccaacaaag gtgaaggtgc acatgaaaaa ggccctgagc tgactcgagt ctagagggcc
     3601 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3661 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3721 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3781 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3841 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3901 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3961 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4021 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4081 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     4141 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4201 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     4261 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4321 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4381 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4441 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4501 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4561 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     4621 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     4681 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4741 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4801 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4861 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     4921 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4981 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     5041 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     5101 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     5161 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     5221 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     5281 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     5341 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     5401 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     5461 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     5521 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     5581 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5641 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5701 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5761 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5821 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5881 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5941 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     6001 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     6061 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     6121 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6181 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6241 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca tag
//
