LOCUS       sequence-446514-        9023 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Used for adding C-terminal SpyTag003 to the E. coli HS P2 phage 
            major capsid protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9023)
  AUTHORS   Ortiz de Ora L, Wiles ET, Zund M, Banuelos MS, Haro-Ramirez N, Suder
            DS, Ujagar N, Angulo JA, Trinh C, Knitter C, Gonen S, Nicholas DA, 
            Wiles TJ
  TITLE     Phollow: Visualizing Gut Bacteriophage Transmission within Microbial
            Communities and Living Animals.
  JOURNAL   bioRxiv [Preprint]. 2024 Jun 13:2024.06.12.598711. doi: 
            10.1101/2024.06.12.598711.
  PUBMED    38915633
REFERENCE   2  (bases 1 to 9023)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Jun 13:2024.06.12.598711. doi: 
            10.1101/2024.06.12.598711."
COMMENT     Sequence Label: pAX1_HS_gpN:SpyTag003
FEATURES             Location/Qualifiers
     source          1..9023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     455..475
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      558..652
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      755..841
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2879..2898)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2998..3020
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3058..3076)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3144..3248
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3249..3317,3318..4109)
                     /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(3467..3486)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5157..5379
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5427..6377
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     oriT            6747..6855
                     /note="incP origin of transfer"
     promoter        join(7119..7124,7125..7141,7142..7147)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             7336..7869
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        7983..8085
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(8152..8171)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     RBS             8807..8818
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(8825..9023,1..515)
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(8855..8883)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
ORIGIN
        1 gtgttcaatg ctttgcgaga tacccagatc atatgaaaca gcatgacttt ttcaagagtg
       61 ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac gggaactaca
      121 agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc gagttaaaag
      181 gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac aactataact
      241 cacacaatgt atacatcatg gcagacaaac aaaagaatgg aatcaaagtt aacttcaaaa
      301 ttagacacaa cattgaagat ggaagcgttc aactagcaga ccattatcaa caaaatactc
      361 caattggcga tggccctgtc cttttaccag acaaccatta cctgtccaca caatctgccc
      421 tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg
      481 ctgggattac acatggcatg gatgaactat acaaataagc gagctcgttt aaacgctagc
      541 ttaattagct gagcttggac tcctgttgat agatccagta atgacctcag aactccatct
      601 ggatttgttc agaacgctcg gttgccgccg ggcgtttttt attggtgaga atccaagcta
      661 gactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa
      721 acgcctgggg taatgactct ctagcttgag gcatcaaata aaacgaaagg ctcagtcgaa
      781 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
      841 tccgccgcta ggagtcgcga gtttaaaccc cggtgttggc agcaccattg ccggaaccac
      901 tgataccacc gcgaaagagc gtgaacctgt cgatccgacg ctgatggtcg atgtggaata
      961 taaatgcgag cagaccaact ttgacacggt gctgacctac gcgaagctgg acctgtgggc
     1021 gaagtttcag gatttccagg tgcgtatccg tgacgccatc gtgaaacgtc aggcactgga
     1081 ccgcatcatg atcggcttta acggcgtgaa gcgtgcgaaa acctccaacc gtagcgaaaa
     1141 cccgctgctg caggatgtga ataaaggctg gctgcagaaa atccgtgagg atgcaccgga
     1201 tcacgtcatg ggcagcacca ccacgggcgg tgaaactaca ccgggtgcgg tgaaagtcgg
     1261 gaaaggtggc gaatatgcca acctggacgc cgtagtgatg gatgccgtca atgagcttat
     1321 cgacgtggtt taccaggacg atgacgatct ggtggttatt tgcgggcgtg aactgctgtc
     1381 tgacaagtat ttcccgctgg tcaacaaaga gcaggaaaac agtgaaaaac tggctgccga
     1441 tatgatcatc agtcagaaac gcatgggtgg cctgcaggcc gtgcgtgcgc cgttcttccc
     1501 gccgaatgcg ctgctaatca cccgtctgga taacctgtcc atctactggc aggaagacac
     1561 ccgccgccgt tcagttatcg acaacccgaa acgtgaccgg attgaaaact ttgaatccgt
     1621 taacgaagcc tatgtggttg aggactaccg ctgcgccgca ctggtggaaa acatccagat
     1681 tggcgatttc agcgccgccg tagcagaaac cggagagtcg ggaggtggtt caggccgtgg
     1741 cgttcctcat attgttatgg tggacgccta caaacgctat aaataatcca tgagcctgag
     1801 tcccgcacgg cagcatcgcc tgcgcgttca ggctgaacag gccgcccgcg agggcggcag
     1861 tgttcgccac gcatcgggct atgacctgat gctgctgcaa ctggcggaag accgccgccg
     1921 tctcaagggc gttcagtcca cggtcaaaaa agcggaaatc aaggtggagc tgctgccgaa
     1981 gtacgccgcc tgggcagagg gtgtcctggc tgccggaggc gctcaacagg atgacgtgct
     2041 gatgtacgtg atgctgtggc gcattgatgc cggagattat gccggggcgc tggagatcgg
     2101 gcgtcatgcc ctgcgtcatg gctgggtgat gccgctgggt aaccgcaacg tgcagaccgt
     2161 gctggcagag gaaatggcag acgccgcgca gagcgcaatg cttgccgcca ccggctttga
     2221 tgccgatctg ttgctgcaga cgctggagct gacagacggt ctggatatgc cggaccagtc
     2281 acgggcgcgt ctgcataaag cgattggcgc tgtcctgagt gaaaacaatc cggcttccgc
     2341 ccttaatcat ctcaaccatg cgttacagct cgatccccgc tgtggcgtga aaaaagacaa
     2401 acagcagctg gagcgcagac tgcgcaatga cagccgctga cagaacgtgc ccccgcgcac
     2461 gggcggcacg gggtggcgaa aggcactgcc acatcaaaac cccgtccacc gccctctatt
     2521 tcaggagaaa gcagcatgaa gtttgttgcg ctagaacagg caccggaaca ggcggaaatc
     2581 atcagaaata cgccgttctg gcctgatgtg gacctgtcgg ggggtcgacc agactcgagt
     2641 ggaggccggc ccctaggacg actgcagccc gcttacgcag ggcatccatt tattactcaa
     2701 ccgtaaccga ttttgccagg ttacgcggct ggtcctctag ctggcgtaat agcgaagagg
     2761 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc
     2821 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta
     2881 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     2941 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3001 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     3061 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     3121 gtggcacttt tcggggaaat gtacgcggaa cccctatttg tttatttttc taaatacatt
     3181 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3241 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3301 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3361 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3421 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3481 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3541 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3601 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3661 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3721 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3781 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3841 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3901 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3961 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4021 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4081 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4141 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4201 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4261 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4321 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4381 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     4441 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4501 tcctgttacc agccggttgt cagccgttaa gtgttcctgt gtcactcaaa attgctttga
     4561 gaggctctaa gggcttctca gtgcgttaca tccctggctt gttgtccaca accgttaaac
     4621 cttaaaagct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     4681 ccgttgatga taccgctgcc ttactgggtg cattagccag tctgaatgac ctgtcacggg
     4741 ataatccgaa gtggtcagac tggaaaatca gagggcagga actgctgaac agcaaaaagt
     4801 cagatagcac cacatagcag acccgccata aaacgccctg agaagcccgt gacgggcttt
     4861 tcttgtatta tgggtagttt ccttgcatga atccataaaa ggcgcctgta gtgccattta
     4921 cccccattca ctgccagagc cgtgagcgca gcgaactgaa tgtcacgaaa aagacagcga
     4981 ctcaggtgcc tgatggtcgg agacaaaagg aatattcagc gatttgcccg attgcggccg
     5041 caaccgagct tgcgagggtg ctacttaagc ctttagggtt ttaaggtctg ttttgtagag
     5101 gagcaaacag cgtttgcgac atccttttgt aatactgcgg aactgactaa agtagtgagt
     5161 tatacacagg gctgggatct attcttttta tcttttttta ttctttcttt attctataaa
     5221 ttataaccac ttgaatataa acaaaaaaaa cacacaaagg tctagcggaa tttacagagg
     5281 gtctagcaga atttacaagt tttccagcaa aggtctagca gaatttacag atacccacaa
     5341 ctcaaaggaa aaggactagt aattatcatt gactagccca tctcaattgg tatagtgatt
     5401 aaaatcacct agaccaattg agatgtatgt ctgaattagt tgttttcaaa gcaaatgaac
     5461 tagcgattag tcgctatgac ttaacggagc atgaaaccaa gctaatttta tgctgtgtgg
     5521 cactactcaa ccccacgatt gaaaacccta caaggaaaga acggacggta tcgttcactt
     5581 ataaccaata cgttcagatg atgaacatca gtagggaaaa tgcttatggt gtattagcta
     5641 aagcaaccag agagctgatg acgagaactg tggaaatcag gaatcctttg gttaaaggct
     5701 ttgagatttt ccagtggaca aactatgcca agttctcaag cgaaaaatta gaattagttt
     5761 ttagtgaaga gatattgcct tatcttttcc agttaaaaaa attcataaaa tataatctgg
     5821 aacatgttaa gtcttttgaa aacaaatact ctatgaggat ttatgagtgg ttattaaaag
     5881 aactaacaca aaagaaaact cacaaggcaa atatagagat tagccttgat gaatttaagt
     5941 tcatgttaat gcttgaaaat aactaccatg agtttaaaag gcttaaccaa tgggttttga
     6001 aaccaataag taaagattta aacacttaca gcaatatgaa attggtggtt gataagcgag
     6061 gccgcccgac tgatacgttg attttccaag ttgaactaga tagacaaatg gatctcgtaa
     6121 ccgaacttga gaacaaccag ataaaaatga atggtgacaa aataccaaca accattacat
     6181 cagattccta cctacataac ggactaagaa aaacactaca cgatgcttta actgcaaaaa
     6241 ttcagctcac cagttttgag gcaaaatttt tgagtgacat gcaaagtaag tatgatctca
     6301 atggttcgtt ctcatggctc acgcaaaaac aacgaaccac actagagaac atactggcta
     6361 aatacggaag gatctgaggt tcttatggct cttgtatcta tcagtgaagc atcaagacta
     6421 acaaacaaaa gtagaacaac tgttcaccgt tacatatcaa agggaaaact gtccataccc
     6481 atgtaaaact taaaacctta gaggctattt aagttgctga tttatattaa ttttattgtt
     6541 caaacatgag agcttagtac gtgaaacatg agagcttagt acgttagcca tgagagctta
     6601 gtacgttagc catgagggtt tagttcgtta aacatgagag cttagtacgt taaacttgag
     6661 agcttagtac gtgaaacatg agagcttagt acgtactatc aacaggttga actgcccatg
     6721 ttctttcctg cgttatcaga gcttatcggc cagcctcgca gagcaggatt cccgttgagc
     6781 accgccaggt gcgaataagg gacagtgaag aaggaacacc cgctcgcggg tgggcctact
     6841 tcacctatcc tgcccggctg acgccgttgg atacaccaag gaaagtctac acgaaccctt
     6901 tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata ccgaaaaaat cgctataatg
     6961 accccgaagc agggttatgc agcggaaagt ataccttaag gaatcccctg ataccgctcg
     7021 ccgcagccga acgaccgagc gcagcgggtc agtgagcgag gaagcggaag agaattccaa
     7081 gatcccctga ttccctttgt caacagcaat ggatcgaatt gacataagcc tgttcggttc
     7141 gtaaactgta atgcaagtag cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac
     7201 gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgtac
     7261 agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt
     7321 tatggagcag caacgatgtt acgcagcagc aacgatgtta cgcagcaggg cagtcgccct
     7381 aaaacaaagt taggtggctc aagtatgggc atcattcgca catgtaggct cggccctgac
     7441 caagtcaaat ccatgcgggc tgctcttgat cttttcggtc gtgagttcgg agacgtagcc
     7501 acctactccc aacatcagcc ggactccgat tacctcggga acttgctccg tagtaagaca
     7561 ttcatcgcgc ttgctgcctt cgaccaagaa gcggttgttg gcgctctcgc ggcttacgtt
     7621 ctgcccaagt ttgagcagcc gcgtagtgag atctatatct atgatctcgc agtctccggc
     7681 gagcaccgga ggcagggcat tgccaccgcg ctcatcaatc tcctcaagca tgaggccaac
     7741 gcgcttggtg cttatgtgat ctacgtgcaa gcagattacg gtgacgatcc cgcagtggct
     7801 ctctatacaa agttgggcat acgggaagaa gtgatgcact ttgatatcga cccaagtacc
     7861 gccacctaac tctagacaat atcgatacct gtgacggaag atcacttcgc agaataaata
     7921 aatcctggtg tccctgttga taccgggaag ccctgggcca acttttggcg aaaatgagac
     7981 gttgatcggc acgtaagagg ttccaacttt caccataatg aaataagatc actaccgggc
     8041 gtattttttg agttgtcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc
     8101 actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt
     8161 cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta
     8221 aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc
     8281 ctgatgaatg ctcatccgga attacgtatg gcaatgaaag acggtgagct ggtgatatgg
     8341 gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc
     8401 tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg
     8461 tgttacggtg aaaacctggc ctatttccct aaaggggtta ttgagaatat gtttttcgtc
     8521 tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac
     8581 ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg
     8641 ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt
     8701 aatgaattac aacagtactg cgatgaacta gtctttggca gtttattctt gacatgtagt
     8761 gagggggctg gtataatcac atagtactgt tcgggtacct agaattaaag aggagaaatt
     8821 aagcatgcgt aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga
     8881 tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg atgcaacata
     8941 cggaaaactt acccttaaat ttatttgcac tactggaaaa ctacctgttc catggccaac
     9001 acttgtcact actttcggtt atg
//
