LOCUS       sequence-423479-        6298 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses equimolar Plasma Membrane- and Endoplasmic Reticulum- 
            targeted split-FAST fragments for Short-range ER-PM Contact sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6298)
  AUTHORS   Cali T
  TITLE     SPLICSFAST-P2A
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6298)
  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            "Unpublished"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-PM-P2A
FEATURES             Location/Qualifiers
     source          1..6298
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     578..597
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(816..920)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(995..1014)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1186..1565
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1566..1769
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1720..1740
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1814..1833
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1814..1832
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1852..1861
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2152..2175
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2203..2259
                     /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             2269..2298
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2840..2857)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2846..3070
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3116..3544
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3203..3222)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3413..3434
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3553..3573)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3558..3887
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3738..3873
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3800..3819
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3954..4748
                     /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(4008..4027)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4618..4637
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4922..5043
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4959..4978)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5013..5032
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5092..5108)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5092..5108)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5105..5127)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5116..5132
                     /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(5140..5146,5147..5164,5165..5170))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5185..5206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5323..5340)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5494..6082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5574..5593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6253..6298,1..746,747..815))
                     /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"
ORIGIN
        1 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
       61 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
      121 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
      181 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
      241 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
      301 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
      361 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
      421 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
      481 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
      541 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
      601 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
      661 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
      721 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
      781 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
      841 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
      901 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
      961 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     1021 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     1081 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     1141 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     1201 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     1261 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     1321 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     1381 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     1441 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     1501 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     1561 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     1621 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     1681 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     1741 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
     1801 ggcttatcga aattaatacg actcactata gggagaccca agctggctag cgccaccatg
     1861 ggcgatagct actgggtgtt tgtgaagcgg gaattcggcg gcgacggcgg atccggaggc
     1921 ggcgatatct ctaagctgcg ggtgttcctg gctctgccta tcatcatggt ggtggctttc
     1981 agcatgtgca tcatctgcct gctgatggcc ggagacacct ggaccgagac cctggcctac
     2041 gtgctgtttt ggggtgtggc cagcatcggc acattcttca ttattctgta caacggcaag
     2101 gacttcgtgg acgcccctag actggtgcag aaggaaaaga tcgacgatat cgactacaaa
     2161 gatgatgatg acaagctgaa ggagctggcc tgtggatccg gcgccaccaa cttcagcctg
     2221 ctgaagcaag ccggcgacgt ggaagagaac cctggtccca gatctatgga gcagaaactg
     2281 atcagcgagg aagatctgtg cggcctgttc atctgtcctt gcaacaagct caaaagcagc
     2341 gacgcttata agaaggcctg gggcaacaac caggacggcg tggtcgcctc tcagccagcc
     2401 agagtgggca gcggcggctc cagcggatct cggggctctg atggcagcga cggctccatg
     2461 gaacacgtgg cctttggaag cgaggacatc gagaatacac tggccaagat ggacgacgga
     2521 cagctggacg gccttgcctt tggcgctatc cagctggacg gcgatggcaa catcctgcag
     2581 tacaatgccg ccgagggcga catcaccggc agagacccca agcaggttat cggcaaaaac
     2641 ttcttcaagg atgtggctcc aggcaccgac agccccgagt tctacggcaa gttcaaggaa
     2701 ggcgtcgcca gcggcaatct gaacacaatg ttcgagtgga tgatccctac aagcagaggc
     2761 cctaccaagg tgaaggtgca catgaaaaag gccctgagct gactcgagtc tagagggccc
     2821 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2881 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2941 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     3001 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     3061 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     3121 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3181 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3241 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3301 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3361 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3421 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3481 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3541 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3601 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3661 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3721 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3781 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3841 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3901 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     3961 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     4021 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     4081 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     4141 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     4201 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4261 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4321 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4381 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4441 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4501 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4561 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4621 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4681 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4741 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4801 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4861 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4921 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4981 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5041 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     5101 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5161 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5221 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5281 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5341 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5401 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5461 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5521 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5581 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5641 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5701 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5761 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5821 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5881 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5941 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6001 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6061 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6121 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6181 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6241 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtcta
//
