LOCUS       sequence-462275-        6980 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of FAM177A1-Halo in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6980)
  AUTHORS   Ugur B, Schueder F, Shin J, Hanna MG, Wu Y, Leonzino M, Su M, McAdow
            AR, Wilson C, Postlethwait J, Solnica-Krezel L, Bewersdorf J, De 
            Camilli P
  TITLE     VPS13B is localized at the cis-trans Golgi complex interface and is 
            a functional partner of FAM177A1.
  JOURNAL   bioRxiv [Preprint]. 2023 Dec 18:2023.12.18.572081. doi: 
            10.1101/2023.12.18.572081.
  PUBMED    38187698
REFERENCE   2  (bases 1 to 6980)
  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: "bioRxiv 
            [Preprint]. 2023 Dec 18:2023.12.18.572081. doi: 
            10.1101/2023.12.18.572081."
COMMENT     Sequence Label: FAM177A1-Halo
FEATURES             Location/Qualifiers
     source          1..6980
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1040..1161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1051..1070)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1105..1124
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1335..2129)
                     /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(1446..1465)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2056..2075
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2196..2525)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2210..2345
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2264..2283)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2510..2530
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2539..2967)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2649..2670)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2861..2880
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3013..3237
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3226..3243
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3263..4153)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     regulatory      4870..4879
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4932..4951)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4933..4951)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4996..5199)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5025..5045)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5200..5579
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5751..5770
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5845..5949
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5950..6018,6019..6810)
                     /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     complement(6168..6187)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1081 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1141 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1201 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1261 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa
     1321 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     1381 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     1441 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     1501 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     1561 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac
     1621 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     1681 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     1741 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     1801 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     1861 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     1921 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
     1981 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     2041 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     2101 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     2161 gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag gcctccaaaa
     2221 aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc ctctgcataa
     2281 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     2341 gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc
     2401 atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga
     2461 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga
     2521 cacacattcc acagaattaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2581 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2641 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2701 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2761 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2821 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2881 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2941 cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag ctggttcttt
     3001 ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg tcttcccaat
     3061 cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac
     3121 aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg
     3181 tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacagtcg
     3241 aggctgatca gcgggtttat cagccggaaa tctcgagcgt cgacagccag cgcgcgatct
     3301 cgctgccgat caggtccggg ttgtcttctt gcagcagatt cagacccggg ccgatgtcca
     3361 cagccttgca gttaggcagg cttttggcca ggcgagcggc ttcggccggt gggatcagaa
     3421 cgcctggggt gccccagaac agcagcttcg ggacagggga ctggtgcagc cagtccatgt
     3481 attcttcgac cagcgcgacg atgttcgctg gctcaccggc gattggcagc tcgtttggga
     3541 agcgccacag tggctcgcgg tcaacaggat tcaggaacgg ctcgcggtaa tggtccatct
     3601 cgacttcagt cagcgggcgg acgacaccca tcggcagcgt accctcgata aaaacgttct
     3661 gatcgatgat cagcttgcgg ccgacgtcgg tggtgcggaa ggcctggaag gtctcgcggg
     3721 caaattctgg ccattcgtcc caggtcggga tagggcggat gaactccata aatgcaatac
     3781 ctttgacgcg ctctggattg cgcttggccc agtggaaacc cagagcggag ccccagtcgt
     3841 gaatgaccag gacgacctct tccagaccca gggcttcgat gaaggcatcc atgaagcgga
     3901 cgtggtcgtc gaagaaataa cccaggtctg gtttgtcgga tttgcccata ccgatcaggt
     3961 ctggagcaat gcagcgatgg gtcggtgcaa catgcgggat gatgttgcgc cacacgtagg
     4021 aggaggtcgg gttaccgtgc aggaacagca caggggtgcc atcgcgcgga ccaacatcga
     4081 cgtagtgcat gcgctcgccc aggacttcca cataatgggg gtcgaatgga aagccagtac
     4141 cgatttctgc cattccactt cctcctggtg ggacagagac tggattttgc ttgctttcca
     4201 taattacttc actgtctccc tccatttcaa aattgacatt cacaaatgag ctggatatca
     4261 ctgtctctgg ttgatctgtc tgaacaatgg aatcagtctg caatttattc tgttgatatt
     4321 gtttttctgc ttcttcagac atcctgtttt cttcttcttc ttcttcttcc tccttcttca
     4381 tccgataata ttcatcaatg gcatattggt actttggggt gctgataccc aaaacagatg
     4441 caatcttctc tccaaggaag tcacacactg agagagttga tgtagcagcc cgaagcatgt
     4501 aaaaccataa gtagggaccc caggtaagtt ttgtcggatc aacagtaggc aaaacatctt
     4561 tcttctccag gccatcaact tcgtcttcat ctgtgctata ttcttccatt gtttcaccac
     4621 taacaaagtg gatgactctc cttgggactt tcttcttttt tcctatgact cccagttcta
     4681 cattttcaaa gcctctttcg ttactcatct gccctgcaga ttccccgaat gccgcggcgg
     4741 ccgcagctcc cgaggctgcg acggcttctc ctcgttccac accgcccact ggctcctggt
     4801 ccatcgtcgt cgccaccaca gggctgctgg cgctggtgag aaagagggta atggccggta
     4861 agcccacttc catggtggcg gatccgagct cggtaccaag cttaagttta aacgctagcc
     4921 agcttgggtc tccctatagt gagtcgtatt aatttcgata agccagtaag cagtgggttc
     4981 tctagttagc cagagagctc tgcttatata gacctcccac cgtacacgcc taccgcccat
     5041 ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag tcccgttgat tttggtgcca
     5101 aaacaaactc ccattgacgt caatggggtg gagacttgga aatccccgtg agtcaaaccg
     5161 ctatccacgc ccattgatgt actgccaaaa ccgcatcacc atggtaatag cgatgactaa
     5221 tacgtagatg tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca
     5281 ggcgggccat ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg
     5341 atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt
     5401 ccctattggc gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt
     5461 gggcggtcag ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg
     5521 gctatgaact aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca
     5581 acgcgtatat ctggcccgta catcgcgaag cagcgcaaaa cgcctaaccc taagcagatt
     5641 cttcatgcaa ttgtcggtca agccttgcct tgttgtagct taaattttgc tcgcgcacta
     5701 ctcagcgacc tccaacacac aagcagggag cagatactgg cttaactatg cggcatcaga
     5761 gcagattgta ctgagagtgc accatagggg atcgggagat ctcccgatcc gtcgacgtca
     5821 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5881 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5941 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     6001 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     6061 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     6121 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     6181 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     6241 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     6301 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     6361 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     6421 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6481 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6541 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6601 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6661 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6721 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6781 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6841 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6901 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6961 gaaaagatca aaggatcttc
//
