LOCUS       sequence-163349-        4059 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes illumina barcodes for allele frequency sequencing 
            (FREQ-Seq-Squared).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4059)
  AUTHORS   Zhao R, Lukacsovich T, Gaut R, Emerson JJ
  TITLE     FREQ-Seq2: a method for precise high-throughput combinatorial 
            quantification of allele frequencies.
  JOURNAL   G3 (Bethesda). 2023 Sep 30;13(10):jkad162. doi: 
            10.1093/g3journal/jkad162.
  PUBMED    37494033
REFERENCE   2  (bases 1 to 4059)
  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: "G3 
            (Bethesda)."; date: "2023-09-30"; volume: "13(10):jkad162. doi"; 
            pages: " 10.1093/g3journal/jkad162"
COMMENT     Sequence Label: TOPO-BC20
FEATURES             Location/Qualifiers
     source          1..4059
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             217..663
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSYLGDTIEYSSYASSLVPSSDPLVTAASVLEFALVSSGF
                     SRLKDRKSGSAGMPRPISYAVFCLRANSADIHHTGGRSSMHLEGPIRPIVSRITIHWPS
                     FYNVVTGKTLALPNLIALQHIPLSPAGVIAKRPAPIALPNSCAA"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(492..511)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(493..511)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(518..535)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(518..534)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(527..549)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      675..1103
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(762..781)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     972..993
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             1447..2241
                     /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(1501..1520)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2111..2130
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(2259..2327,2328..3119)
                     /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(2477..2496)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3290..3878
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3779..3798
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4032..4049
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gcccttgtaa gcagtgggtt ctctaggctt
      361 aaagatcgga agagcggttc agcaggaatg ccgagaccga tctcgtatgc cgtcttctgc
      421 ttaagggcga attctgcaga tatccatcac actggcggcc gctcgagcat gcatctagag
      481 ggcccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc
      541 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
      601 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
      661 tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
      721 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
      781 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
      841 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
      901 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
      961 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     1021 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     1081 ttaacaaaaa tttaacgcga attttaacaa aattcagggc gcaagggctg ctaaaggaag
     1141 cggaacacgt agaaagccag tccgcagaaa cggtgctgac cccggatgaa tgtcagctac
     1201 tgggctatct ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg
     1261 cttacatggc gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca
     1321 gctggggcgc cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg
     1381 ccgccaagga tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg
     1441 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     1501 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     1561 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     1621 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     1681 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     1741 gggcaggatc tcctgtcatc ccaccttgct cctgccgaga aagtatccat catggctgat
     1801 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     1861 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     1921 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     1981 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     2041 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     2101 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     2161 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     2221 cttcttgacg agttcttctg aattgaaaaa ggaagagtat gagtattcaa catttccgtg
     2281 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     2341 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     2401 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     2461 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     2521 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     2581 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     2641 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     2701 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     2761 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     2821 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     2881 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     2941 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     3001 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     3061 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     3121 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     3181 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     3241 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3301 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3361 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3421 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3481 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3541 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3601 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3661 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3721 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3781 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3841 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3901 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3961 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4021 cgaccgagcg cagcgagtca gtgagcgagg aagcggaag
//
